OF PIGLETS,
DIETARY PROTEINS,
AND PANCREATIC PROTEASES
Promotor:
Dr Ir M.WA. Verstegen
buitengewoon hoogleraar op het vakgebied van de veevoeding,
in het bijzonder de voeding van de eenmagigen
Co-promotor:
Dr Ir LA. den Hartog
directeurvanhetproefstationvoordevarkenshouderij te Rosmalen
lOMOP
OF PIGLETS,
DIETARY PROTEINS,
AND PANCREATIC PROTEASES
Caroline A. Makkink
Proefschrift
ter verkrijging van de graad van
doctor in de landbouw- en milieuwetenschappen,
op gezag van de rector magnificus,
dr H.C. van der Plas,
in het openbaar te verdedigen
op donderdag 3juni 1993
des namiddags te vier uur in de aula
van de Landbouwuniversiteit te Wageningen
OOOOi
U K _.^S o Lo
1
/. lo3<i
VOORWOORD
Dit proefschrift is het resultaat van 4V2jaar onderzoek bij de vakgroep Veevoeding. In
die 4Vijaar vol mee- en tegenvallers ismij duidelijk geworden dat steun en gezelligheid
van collega's onontbeerlijk zijn voor het behoud van 'lol inje werk'. Gelukkig heeft het
hieraan bij Veevoeding nooit ontbroken.
Enige mensen, die, elk op hun eigen wijze, een bijdrage hebben geleverd aan de
totstandkoming van dit proefschrift, wil ik met name noemen:
Allereerst natuurlijk mijn promotor, prof, dr ir M.W.A. Verstegen. Martin, jouw
suggesties en ideeen (hoewel vaak onverstaanbaar of onleesbaar) bleken (na
ontcijfering) altijd erg waardevol te zijn. Vat het niet persoonlijk op dat ikje tips voor
stellingen ("Melk moet mogen") niet heb benut.
Dr ir L.A. den Hartog, mijn co-promotor. Leo, jij bent vanaf het begin betrokken
geweestbijhet onderzoek enjewasvanonschatbarewaardebijhet nategenslagenweer
'op de rails zetten' van mij en m'n onderzoek.
Toen Leo vertrok naar Rosmalen nam dr ir B. Kemp veel van zijn klussen over,
waaronder mijn begeleiding.Bas,jouwfelle inbreng,vooral op het statistischevlak heeft
mij veel verder geholpen. Uit resultaten, waar ik vaak kop noch staart aan kon
ontdekken, wist jij altijd listige theorieen te destilleren.
Ook Basverliet de vakgroep na enige tijd (zou het aan mij liggen?) en werd opgevolgd
door dr ir M.W.Bosch.Marlou,jij hebt de laatste fases van het onderzoek meegemaakt
en daarmee waarschijnlijk ook de leukste discussies. Jouw praktische en concrete
instelling heeft me erg aangesproken, bovendien had dit proefschrift zonder jouw inzet
waarschijnlijk een hoofdstuk minder geteld...
Bij de proeven met gecannuleerde biggen heeft het ILOB een grote rol gespeeld. Met
nameJoopHuisman, PietvanLeeuwen, DickvanKleef enKasperDeuringwilik hierbij
hartelijk danken voor hun hulp. Martin van Baak en zijn lab-medewerkers hebben veel
werk verricht om de enzymanalyses van de grond te krijgen. Ook Martje Fentener van
Vlissingen heeft door haar bemoeienissen een belangrijke bijdrage geleverd aan mijn
ontwikkeling en aan dit proefschrift.
De medewerkersvanproefaccomodatie De Haarwaren altijdbereid meetedenken met
de praktische invullingvan de proeven. De inzetvan Jan Hagens, Ries Verkerk, Andre
Jansen en de anderen heb ik erg gewaardeerd.
Mijn paranimfen, Josienvan de Kraats enAnnemarie Zijlmans waren altijd beschikbaar
voor een praatje en een pafke, zodat ik mijn kleine frustraties en irritaties weer snel
kwijt raakte.
Ook alleandere medewerkers van devakgroep hebben hun steentje bijgedragen aan dit
proefschrift door hun assistentie bij het slachten van ca 150 biggen en door hun
aandacht en steun in voor- en tegenspoed. Bedankt hiervoor!
MiSLLOillLhlv
r"ANn»OU\VUMVERSITEIT
v :
* u;r.NiM;EN
1
no
fJtJOt
y
STELLINGEN
I
Verschillen in schijnbare verteerbaarheid tussen ondermelkpoeder, sojaprodukten en
vismeel bijjonge biggen zijn voornamelijk te wijten aan verschillen in endogene stikstofverliezen.
(dit proefschrift)
II
De exocriene pancreas is niet de belangrijkste bron van endogene stikstof-verliezen bij
jonge biggen.
(dit proefschrift)
III
Onvoldoende overeenstemming met betrekking tot monster-bewaaromstandigheden en
enzymanalyses bemoeilijkt de interpretatie van literatuur-gegevens.
(dit proefschrift)
IV
Bijjonge biggen is de voeropname na het spenen belangrijker dan de eiwitbron in het
voer voor de ontwikkeling van pancreas-protease-activiteiten.
(dit proefschrift)
Het door Szabo et al (1976) beschreven effekt van omgevingstemperatuur op (pancreas)
enzymactiviteiten bij ad lib gevoerde varkens moet waarschijnlijk geinterpreteerd worden
als een effekt van voeropname.
(Szabo et al. (1976) Magyar Allatorvosok Lapja 31(5):325-328)
(dit proefschrift)
i(_af>:l
VI
"Het kweken van tomaten en het fokken van varkens gebeurt in Nederland op meer
wetenschappelijke basis dan het voeren van overheidsbeleid".
(A. Hoogerwerf, bestuurskundige TU Twente, in 'NOVA', 19maart 1993)
VII
De theorie, dat bepaalde karaktereigenschappen de kans op het krijgen van (ernstige)
ziektes kunnen vergroten, isvooral op humane gronden verwerpelijk.
VIII
Positieve discriminatie van vrouwen bij sollicitaties leidt tot discriminatie van mannen die
een baan zoeken en tot discriminatie van vrouwen die een baan hebben.
IX
Het bezit van kennis zonder het vermogen tot kennisoverdracht is nutteloos.
X
Proefdieren schijnen te streven naar maximale variatie binnen proefgroepen, waardoor
veel dierfysiologische experimenten, gericht op verschillen tussen proefbehandelingen in
de war gestuurd worden.
Stellingen behorende bij het proefschrift van C.A. Makkink, getiteld 'Of piglets, dietary
proteins, and pancreatic proteases'.
Wageningen, 3juni 1993
Fur das 16N-Experimentbeschrieben inKapitel IIwarender personliche Einsatzund die
Begleitung von Dr Th. Heinz und Dr W.B. Souffrant fur mich von groBem Belang.
Vielen Dank dafur!
I alsowant to acknowledge thework of dr George Puia Negulescu from Roumania and
Dr Qin Gubtin from China who participated as guest workers. Niki and Qin, I really
enjoyed working with you, we were a great team!
Natuurlijk waren ook de studenten en stagiaires onmisbaar voor het welslagen van de
proeven.Involgordevan opkomst:LucettevanderWesterlaken,Petravan Oosterbosch,
Nancy Nieuwenboom, Sander Huitink, Piet Donkers, Pierre Berntsen, Brigitte op den
Kamp en Rody de Wolf. Bedankt voor jullie inbreng en betrokkenheid.
Al met al kijk ik met veel plezier terug op mijn AlO-tijd, en daarvoor bedank ik
iedereen (al dan niet met name genoemd) die daarbij betrokken was.
Carolien
OP EEN BIG
EEN BRITSE BIG WIENS NAAM WAS PORK
LIEP ALTIJD ROND MET MES EN VORK.
"IK DRAAG DIT BIJ MIJ", SPRAK HET BEEST,
"ZOALS EEN IEDER, DIE DIT LEEST
EN BRITS VERSTAAT, TERSTOND ZAL SNAPPEN,
FOR IF THE WORST
SHOULD COME TO
HAPPEN".
(TRIJNTJE FOP)
CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG
Makkink, Caroline A.
Of piglets, dietary proteins, and pancreatic proteases /
Caroline A. Makkink. - [S.l. : s.n.]
Proefschrift Wageningen. - Met lit. opg. - Met samenvatting in het Nederlands.
ISBN 90-5485-104-X
Trefw.: varkens ; voeding
Makkink,C.A., 1993.Ofpiglets,dietaryproteins,andpancreaticproteases (Vanbiggen,
voereiwitten en pancreas proteases).
Newly weaned piglets often show digestive disorders, frequently resulting in diarrhoea.
These disordersmaybe related tothe dietaryprotein source,sinceyoungpigletsare less
capable of digesting proteins of vegetable origin than older pigs. This study was
undertaken to investigate the development of protein digestive capacity in weaned
piglets. In the first part of the thesis it was established that the difference in nitrogen
digestibility between milk, soya and fish proteins was mainly due to differences in
endogenous nitrogen losses and not to differences in true digestibility. In the second
part, the pancreatic protease activities of newly weaned piglets fed various dietary
protein sourceswasinvestigated.Itwasfound that dietaryprotein sourceaffected trypsin
and chymotrypsin activities in pancreatic tissue and jejunal digesta, however, these
differences could not explain the differences in endogenous nitrogen losses. It is more
likely that other sources of endogenous nitrogen, e.g. the small intestinal wall, are
responsible for the differences in protein digestibility observed in young piglets.
Furthermore, it was concluded from the experiments described in this thesis, that postweaning feed intake has a greater effect on the development of pancreatic enzyme
activities than dietaryprotein sourceperse.Youngpigletsare capable of increasing their
pancreatic secretion in response to appropriate stimuli. Stimulating post-weaning feed
intake may help to smooth the development of protein digestive capacity in young
piglets. In this respect, feed intake patterns maybe at least as important as quantitative
feed intake.
Ph.D. thesis.Department of Animal Nutrition,Agricultural University, Haagsteeg4,6708
PM Wageningen,TheNetherlands.
CONTENTS
page
General Introduction
II
Endogenous N Losses at the Terminal Ileum
of Young Piglets Fed Diets Based on
Four Different Protein Sources
29
III
Pancreatic Secretion in Pigs
45
IV
Storage of Porcine Pancreatic Juice:
Effect on Enzyme Activities
75
Gastric Protein Breakdown and Pancreatic
Enzyme Activities in Response to Two Different
Dietary Protein Sources in Newly Weaned Piglets
85
Effect of Dietary Protein Source on Feed Intake,
Growth, Pancreatic Enzyme Actvities and Jejunal
Morphology in Newly Weaned Piglets
103
A Note on the Response of Pancreatic Juice Secretion
to Different Stimulants in Young Pigs
123
V
VI
VII
VIII General Discussion
131
Summary
157
Samenvatting
161
List of Publications
165
Curriculum vitae
168
ChapterI
GENERAL INTRODUCTION
Chapter I
General
The first food that a newborn piglet ingests is colostrum from the sow. Until weaning
milk is the major source of nutrients. Young piglets are very well capable of digesting
nutrients from colostrum and milk immediately after birth.
Half a century ago, piglets were normally weaned between 8 and 12 weeks of age
(Linton, 1927),graduallyincreasingtheirsolidfeed intake.Nowadays,weaninghasmoved
to an earlier age (3 to 4weeks) so as to increase sow productivity. Developments in the
preparation ofweaner diets (milkreplacers) have enabled thepiglet tobeweaned at this
age.
However, after weaning at 3to 4weeks of age,growth checksand digestive disorders are
often experienced in modern piglet rearing. The causes of these problems concerning
newlyweaned piglet performance are many-fold. Weaning includesvarious stress factors
for the young piglet. The change to a new environment imposes climatic stress factors
upon theyoungpiglet.Especially the nutritional challenge isa crucial aspect ofweaning.
The withdrawal of sows milk is an important factor in the process of weaning, not only
from a nutritional point of view (composition of sows milk with respect to easily
digestible nutrients) but also in a behavioral and immunological sense (Stanton and
Mueller, 1976).
The piglet must learn to consume a solidweaning diet of a completely different texture,
temperature, smell, taste and composition compared to sows' milk.
Protein isthe most expensive nutrient inpigfeeds, especiallywhen milkproteins are fed.
Because milkproducts willbe less available inthe future, attempts are made to decrease
the proportion of milk proteins and increase the amount of vegetable protein sources in
post-weaning pig diets. The incorporation of large amounts of non-milk proteins,
however, often leads to digestive disorders in young piglets (Cline, 1991).
The adaptation of the piglet's digestive system to different protein sources is a very
complex process. Adaptation must occur with respect to pH regulation (acid secretion
in the stomach and alkaline secretions from the pancreas, gall-bladder and small
intestine), enzyme secretions (stomach, pancreas, small intestine), motility (gastric
emptying, passage rate of digesta in different parts of the gastrointestinal tract) and
absorption (transport mechanisms) (Schnabel, 1983;Seve, 1985; Cline, 1991).
The digestion of the diet depends not only on dietary factors (energy/protein content,
antinutritional factors, (non)essential amino acids) but also on factors concerning the
animal that consumes the diet (age, breed, weaning circumstances, health status, sex).
Environmental factors (temperature, humidity, hygiene) also play a part indigestion and
performance.
10
Chapter I
In current practice, the piglets are introduced to creep feed from one or two weeks of
age onwards. Creep feed isusually based on milk proteins and contains easily digestible
carbohydrates and fats.
About one week before, or sometimes just after weaning, other protein sources are
incorporated inthe piglets'diet.The shift from milkprotein to other dietary proteins can
put too great a strain on the piglets'immature digestive system. This can lead to growth
depression and/or (post weaning) diarrhoea.
Digestionof DifferentProteinSourcesin Piglets of DifferentAges
Protein digestion is usually measured as apparent faecal nitrogen digestion. Especially
with newlyweaned piglets,not many data are available on ileal nitrogen digestibility, let
alone on true nitrogen digestibility. Individual housing and intestinal surgery are more
difficult for young piglets than for growing swine and this complicates ileal and true
digestibility studies in piglets.
In thisthesis the term 'digestibility'willapply to apparent faecal digestibility unless stated
otherwise.
In young piglets, the difference in digestibility values between vegetable and animal
protein is considerable. Soy proteins, e.g., are not digested to the same extent as milk
proteins (Wilson and Leibholz, 1981c).Later in life, pigs are more capable of digesting
protein sources from plant origin (Combs etal, 1963;Van de Kerk, 1982).The fact that
the difference in digestibility between animal protein and vegetable protein sources
decreases with advancing age of the piglets (Table 1) leads us to believe that the
'developmental or adaptational state' of the piglet is the bottle neck in the digestion of
protein. Ifwewant to look into the causes of depressed digestion more closely,we have
to consider the process of protein digestion in more detail.
There are different sites inthe gastrointestinal tract of the pigwhere problems can occur
when overall protein digestion isdisturbed. Firstly,feed intake can be depressed as often
occurs duringthe early post-weaning period. Feed intake isaffected bypalatability of the
feed, texture of the feed (pellets, meal, slurry), physical properties (pellet size and
hardness) and generally byfamiliarity of the feed to the animal and bywell-being of the
animal.
Also, the interaction between the solid diet and the piglet's digestive tract is important:
the physical and chemical characteristics ofthe post-weaning diet mayaffect the integrity
of the small intestinal mucosa leading tovillus atrophy and malabsorption. This gut wall
damage may then lead to depressed feed intake.
11
Chapter I
Animportant roleinprotein digestion isplayed bythe stomach.In the stomach,acid and
proteolytic enzymes are added to the feed and clot formation occurs depending on the
type of ingested protein. Gastric emptying patterns determine the release of gastric
contents into the small intestine and thus affect the intestinal digestion of protein.
In the duodenum, pancreatic juice and bile are added to the chyme. Pancreatic juice
containsbicarbonate,whichserves tobuffer the acid contained inthe digesta leaving the
stomach.The intestinal pH needs tobe elevated because the optimum pH for activityof
thepancreaticproteasesliesaround 8.Thepancreaticproteases continuethe breakdown
ofproteinswhichwasinitiated inthestomach.Pancreaticenzymesynthesis,secretion and
activation should respond tothe substrates (e.g.,typeof dietaryprotein) presented inthe
digesta so that adequate digestion of nutrients is ensured.
The protein digestion by intestinal peptidases in the small bowel coincides largely with
absorption since many intestinal proteases are attached to the gut wall so that products
of hydrolysis can be absorbed directly. An intact gut wall mucosa is of course a
prerequisite for undisturbed absorption of nutrients. Gut wall damage is a common
finding during the early post-weaning period in pigs (Nabuurs, 1991).Villus atrophy and
crypt hyperplasia hamper digestion and absorption inthesmallintestine of newlyweaned
piglets.
A disturbed digestion and absorption leads to an excess of nutrients in the lower small
intestine. This may lead to serious alterations in the pig's microflora, resulting in
proliferation of pathogens and probably digestive disorders.
Thus, it is clear that the causes of inadequate protein digestion can be many-fold, since
the digestion of protein is the result of many processes occurring during the passage of
food through the digestive tract. These processes cannot be considered independently
since their interrelationships form the basis for proper protein digestion.
In the next paragraph a description is given of the process of protein digestion in the
digestive tract of the pig.
12
Chapter I
Table 1
ref.
10
11
12
13
14
15
16
Apparent faecal digestibility (protein/N) of diets based on different protein
sources in young piglets.
age (weeks or days)
or weight (kg)
of the piglets:
at
during
weaning exp.
protein source:
skimmed
soya- soya isolated
milk casein bean protein soya
powder
meal cone, protein
fish
meal
15 d.
15 d.
15 d.
8d.
3d.
3d.
21 d.
4-5 d.
4-5 d.
4-5 d.
79.1
81.6
85.7
70.7
69.6
83.7
21 d.
21 d.
5 wks
?
21 d.
21 d.
7d.
7d.
7d.
10 d.
10 d.
21 d.
5-11
5-11
5-11
5-11
5-11
5-11
5-11
5-11
2d.
21 d.
21 d.
3-4 wks
5-6 wks
7-8 wks
25-29 d.
25-29 d.
25-29 d.
3-5 wks
28 d.
14 d.
35 d.
4.7 kg
28 d.
41 d.
5-7 wks
20-22 kg
4 wks
8 wks
2 wks
4 wks
6 wks
18-23 d.
38-43 d.
32-35 d.
39-42 d.
46-49 d.
53-56 d.
32-56 d.
12-16 d.
16-20 d.
20-24 d.
24-28 d.
28-32 d.
32-36 d.
36-40 d.
12-40 d.
23-51 d.
10-14 d.
25-35 d.
42 d.
73.8
75.9
85.0
94.47
93.97
94.19
86.3
98.6
96.8
98.3
82.3
82.2
93.7
95.7
83
80
86
91.75
89.1
79.7 83.6
72
78
76
71.42
88.12
65.64
69.23
74.58
89.89
95.72
68.27
88.16
89.22
95
96
96.09
96.35
95.91
93.96
95.57
93.0
94.6
92.2
92.1
94.0
93.1
95.5
93.5
93.7
95.6
92.61
77
83
93.20
93.15
89.96
94.24
93.63
74.0
83.7
80.06 90.69
76.2 80.8
90.95
80.6
References: l=Comba et al. (1963), 2=Watkins &Veum (1986), 3=Etheridge et al. (1984), 4=Wilson &Leibholi (1981c),
5=Li et al. (1989),6=Christison & ParradeSolano (1982), 7=Moughan et al. (1989), 8=Green &Kiener (1989), 9=Pekas
et al. (1964), 10=Hays et al. (1959), ll=Sewell & West (1966), 12=Decuypere et al. (1981), 13=Sherry et al. (1978),
14=Zamora et al. (1979), 15=Sohn &Maxwell (1990), 16=Walker et al. (1986)
13
Chapter I
The Course of Protein Digestion
A simplified diagram of protein digestion in monogastrics is given in Figure 1.
DIGESTION
HC1
gastric
proteases
HC0 3
pancreatic
proteases
intestinal
peptidases
ABSORPTION
FERMENTATION
Figure 1.
A schematic overview of the sites of digestion and absorption of protein in the
digestive tract of pigs. O=0esophagus, S=Stomach, P=Pancreas, L=Liver,
G=Gallbladder, D=Duodenum, J=Jejunum, I=Ileum, Ca=Caecum, Co=Colon.
The digestion of protein is accomplished by the action of different enzymes along the
gastrointestinal tract. An overview is given in Table 2.
14
ChapterI
Table2
Proteolytic enzymessecreted from different sitesalongthe gastrointestinal tract.
site
enzyme
mouth/saliva 1 stomach
2
chymosin
pepsin
gastricsin
pHoptimum specificity
-
-
6.3
3.5
6.3
2.0
milk clotting
proteolysis
milk clotting
proteolysis at N-side of LEU, ASP,
GLU, PHE, TYR and TRP,
not at C-side of PRO
milk clotting
proteolysis
6.3
3.0
duodenum 3 ' 4,6 trypsin
7.5-8.5
(pancreas)
chymotrypsin 8
small
intestine6
(gut wall)
proteolysis at C-side of LYS,ARG (and AECYS),
not at N-side of PRO
proteolysis at C-side of PHE,
TRP, TYR, MET, GLU, ASP and LEU,
not at N-side of PRO
proteolysis at N-side of C-terminus (PHE, TYR,
TRP, ARG and LYS)
proteolysis at C-side of ALA and GLY
(aliphatic amino acids)
carboxypeptidases
elastase
7.5-8
endopeptidases
aminopeptidase
carboxypeptidases
7.0-7.5
proteolysis, near hydrophobic aminoacids
7.0-7.5
proteolysis, neutral amino acids at N-terminus
7-9
proteolysis, mainly PRO, at C-terminus
7.5-10.5
Kidder and Manners, 1972;Low and Zebrowska, 1989
Sangild, 1990
3
Rinderknecht, 1986; Kidder and Manners, 1972;Wood et al.. 1974
4
Walsh, 1970
5
Wilcox, 1970
6
Rerat and Corring, 1991
2
AccordingtoKidder and Manners (1972)and Lowand Zebrowska (1989)pigsaliva does
not contain any proteolytic enzymes. Protein digestion in pigs starts in the stomach
through the combined action of acid and proteolytic enzymes. Young (suckling) piglets
secrete onlysmall amounts ofpepsinogen and hydrochloric acid (Sangild, 1990).Initially,
the only protein source for the suckling piglet is sow's milk. Gastric digestion of milk
proteins is a specific process, characterized by the clotting of the milk (casein) by
chymosin (rennin) action. Non-milk proteins willnot coagulate in the stomach although
some acid-induced precipitation may occur. Milk (lactose) is also fermented by
15
Chapter I
lactobacilli in the young piglet's stomach, resulting in lactic acid accumulation (Cranwell,
1990).
An undisturbed gastric function depends on animal factors (acid and enzyme secretion,
gastric emptying) and on aspects of the feed (buffering capacity, bulkiness, solubility, clot
formation). These factors cannot be considered separately because they interact, e.g., a
high dietary buffering capacity increases the requirements for acid secretion.
The digestion of protein is continued by the proteolytic action of en2ymes secreted by the
pancreas. T h e p H of intestinal contents is raised through the secretion of HCO s " by the
pancreas. The secretion of pancreatic juice is regulated by nervous and endocrine
mechanisms (Solomon, 1987).
Some hormones and peptides affecting pancreatic secretion are listed in Table 3.
Table 3
Some hormones and peptides affecting pancreatic secretion (Solomon, 1987;
Langlois et al., 1989; Brannon, 1990;Croom et al., 1992)
hormone/peptide
effect
stimulus
secretin
stimulates secretion of
fluid and bicarbonate
unbuffered H +
in duodenum
cholecystokinin-PZ
stimulates secretion of
proteases and growth of pancreas
intraluminal peptides, H + ,
fatty acids, cations,
trypsin inhibitors
pancreatic polypeptide
decreases pancreatic enzyme and
bicarbonate output,
no effect on juice flow
gastrin
stimulates secretion of enzymes
chymodenin
stimulates chymotrypsinogen secretion
?
(entero)glucagon
inhibits pancreatic secretion
?
vasoactive intestinal peptide
stimulates secretion of bicarbonate
somatostatin
decreases gastrointestinal tract
motility, blocks HC1and bile
secretion, inhibits absorption
16
stomach distention,
intragastric peptides or
amino acids (?)
vagus nerves (+)
vagus nerves (-)
splanchnic activity (+)
Chapter I
Secretin and CCK-PZ (cholecystokinin-pancreozymin) are themost important hormones
in the regulation of pancreatic secretion (Solomon, 1987; Croom et al, 1992). The end
products of pancreatic protein digestion are mainly peptides.
It isgenerally accepted that activation of pancreatic zymogens occurs through the action
of enterokinase secreted by the duodenal wall and of trypsin from the pancreas itself.
The presence of end products of pancreatic protein digestion in the intestinal lumen is
recognized and communicated to the exocrine pancreas through various hormones and
peptides. Feedback mechanisms are very important in pancreatic regulation but the
precise (inter)actions of various hormones and peptides remains to be unravelled.
Small intestinal digestion of peptides is performed by the action of brush-border
peptidase enzymes (Low and Zebrowska, 1989). Absorption occurs simultaneously
because digestion is mainly established through peptidases attached to the intestinal
epithelium.Peptides canbe hydrolysed at theluminalsiteand transported asamino acids
or absorbed as peptides and hydrolysed in the cytoplasm of the enterocyte or absorbed
as peptides and transported into the circulation as such (Friedrich, 1989).
Nutrientsthat remainundigested and/or unabsorbed untiltheterminalterminal ileumare
subjected to fermentation byintestinal microbes.Excessive bacterial activity maylead to
formation of toxins and to proliferation of pathogenic bacteria.
Animal FactorsAffecting Protein Digestion
1. pH
The pH in the different parts of the digestive tract influences the course of digestion of
nutrients in pigs and other animals.
Precursors of digestive enzymes are activated at specific pH-values. Digestive enzymes
have specific pH-optima at which their activity is highest (Table 2). Gastric proteolytic
enzymes have two pH-optima: 6.3 for milk-clotting and 2.0-3.5 for proteolysis (Table 2).
The pH in the digestive tract also affects microflora proliferation and thereby
fermentation of feed components (Banwart, 1981;Cranwell, 1990).
Appropriate regulation of pH in the gastrointestinal tract is a prerequisite to ensure
optimal digestion of dietary protein and other nutrients.
The maintenance of an optimal pH in a certain part of the tract depends on the
buffering capacity of the feed or chyme entering that part of the tract and on the
secretory capacity of thepH regulating organs (stomach,pancreas, liver,small intestine).
17
Chapter I
2. Enzyme secretion
The synthesis and secretion of proteolytic enzymes needs to be sufficient to digest the
amount and type of dietary protein offered to the animal. In the stomach of suckling
piglets, the clotting of milk-casein is an important aspect of digestion. Milk clotting
influences gastric emptying and thus ensures a more regular release of nutrients into the
gut (Sangild, 1990).Other protein sources (soya,fish) are hydrolysed differently: they do
not coagulate in the stomach and therefore their digestion probably depends more on
pancreatic enzymes (Pekas et al, 1964). Gastric proteolysis is low in young piglets and
increases with advancing age (Leibholz, 1981,1986).
During suckling, exocrine pancreatic secretion remains lowand isnot stimulated bymilk
ingestion (Pierzynowski, 1991). After weaning, the development of pancreatic enzyme
secretion isless clear: Some authors (Lindemann etal, 1986;Owsleyetal, 1986) report
an initial decline in post-weaning trypsin and chymotrypsin activities in pancreatic tissue
and intestinal contents, presumably caused by 'post-weaning stress' or by low postweaning solid feed intake. Others (Pierzynowski, 1991; Efird et al, 1982) find a
substantially higher pancreatic enzyme secretion or activity in the small intestine from
weaning onwards. Pierzynowski (1991) also found that the ingestion of solid feed after
weaning increases pancreatic secretion while milk ingestion during the suckling period
doesnot affect basalsecretion.The effect ofpost-weaningfeed intake (level and pattern)
on pancreatic enzyme synthesis and secretion is insufficiently clear yet.
3. Motility
The motility of the digestive tract is an important factor in digestion. Stomach emptying
and intestinal passage rate are affected byfrequency of feeding (Braude etal, 1970) and
by dietary protein source (probably through clotting properties) (Maner et al, 1962;
Newport and Keal, 1983).
In pre-ruminant calves the composition of the diet is associated with small intestinal
motility patterns and diarrhoea (Duvaux, 1985). Feeding of cows milk resulted in a
normal motility pattern and no diarrhoea. Soy protein feeding resulted in diarrhoea and
abnormalmotilitypatterns (increased numberofregularspikingactivity(RSA)episodes).
When milk was fed in combination with sucrose, calves developed diarrhoea and the
number of RSA's decreased while long periods of irregular spiking activity (ISA) were
found (Duvaux, 1985).
A relation between feed intake patterns and intestinal motility in newly weaned piglets
has been proposed by Sissons (1989) but experimental proof is still lacking.
18
Chapter I
4. Absorption
The intestinal absorption of protein hydrolysis products has recently been reviewed by
Webb (1990). He concludes that small peptides are absorbed from the small intestine
more rapidly than free amino acids.
Temporary changes in intestinal wall structure (decrease in villus length and increase in
crypt depth) and malabsorption are common findings in newly weaned pigs (Hampson
and Kidder, 1986;Lofstedt, 1986;Nabuurs, 1991).Thevillusatrophy leads to an increase
in endogenous nitrogen losses and to malabsorption. The causes of villus atrophy and
crypt elongation are not sufficiently clear yet. Pre- and post-weaning feeding practices
maybe associatedwithchangesingutwallmorphology.Creepfeeding resulted in deeper
crypts and partly prevented the shortening of villus and the decreased absorption after
weaning (Nabuurs, 1991). Deprez et al. (1987) managed to reduce post-weaning villus
atrophy in newly weaned piglets by providing liquid (slurry) in stead of dry feed.
Relations between gutwall morphology and feed intake of newlyweaned piglets are not
established conclusively yet.
It is clear that animal factors are of major influence on protein digestion. The different
factors mentioned in the foregoing paragraph are affected by the physiological status of
the pig (age, stress, health and disease).
DietaryFactorsAffecting Protein Digestion
1. Buffering Capacity of Feedstuffs
The buffering capacity of feedstuffs is important for the pH regulation in the stomach.
The pH in the stomach should reach low values (2 to 4) to promote pepsinogen
activation and to inhibit bacterial growth. The stomach should excrete enough
hydrochloric acid to provoke this decrease of pH. Certain feedstuffs, especially protein
sources and mineral mixtures,require large amounts of acid toreach lowpH values (see
Table 4).
If the stomach is deficient with respect to acid secretion, highly buffering agents in the
feed should be avoided.
In young animals, the lowering of pH in the stomach is supported by lactic acid
production by bacteria present in the stomach. Lactic acid bacteria grow on lactose,
which is an important ingredient of (sows) milk. This is one of the reasons why milk
products are well digested by young piglets, despite of their high protein content (and
thus high buffering capacity).
19
Chapter I
Table4
Buffering capacity of different feedstuffs (mmol HC1 consumption required to
reach pH4per 100goriginal matter).(Bolduan etal., 1988)
feedstuff
skim milk (acid)
skim milk (fresh)
skim milk (dried)
wheat
barley
yeast
soyabean meal (extracted and toasted)
fish meal
mineral mixture
starter for weaner piglets
buffering capacity
3.07
7.12
66.37
8.99
9.97
30.10
50.68
60.38
1260.50
30.00
In the stomach of unweaned piglets, enzymes are secreted which induce the clotting of
milk-casein (Sangild, 1990).
When the buffering capacity of the feed is too high, or when the acid production in the
stomach istoo low, pH inthe stomachwillremain high, causing proliferation of bacteria
and insufficient activation of pepsinogen. Thus, the digestion of non-milk protein will be
depressed and bacteria can travel through the digestive tract exerting deleterious effects
further down the gut.
To prevent overburdening ofthe stomachduringdifficult periods(e.g.just after weaning)
diets should be formulated with relatively low buffering capacities or with organic acids
added (Bolduan et al, 1988). Increasing the frequency of feeding can also prevent
digestive problems in the stomach.
2. Protein structure
The structure of a protein can be described in terms of primary, secondary, tertiary and
quaternary structure (Pomeranz, 1991).
- primary structure: amino acids composition and sequence.
- secondary structure: spatial configuration by hydrogen
bonds.
- tertiary structure: three-dimensional organization by
disulphide bonds, hydrophobic bonds, hydrogen bonds between side chains,
electrostaticinteractions,dipole-dipoleinteractions,andcharge-dipoleinteractions.
- quaternary structure: aggregation of protein subunits.
The amino acid composition of the protein determines its susceptibility to enzymatic
degradation, as illustrated in Table 2.
20
Chapter I
The structure and spatial organization of a protein determines its functional properties
(solubility,stabilityover pH and temperature ranges) andtherefore affects itsdigestibility
(solubility, accessability towards enzymes).
3. Antinutritional factors
Antinutritional factors (ANF's) can affect the digestion of nutrients through various
mechanisms. Trypsin inhibitors bind to trypsin and thus inhibit the digestion of protein
byremoving active trypsin from the gut,bysuppressing the activation of other zymogens
and by increasing the amount of endogenous nitrogen losses. Tannins bind to protein
moleculesthuspreventing thedigestion oftheseproteins.Lectinsbindtothegutwall and
hamper absorption of digestive end products. The effects of ANF's on dietary protein
utilization have recently been summarized by Nitsan (1991).
4. Interactions between nutrients
A nutrient may interfere with the digestion of other nutrients inthe diet. Carbohydrates
are known to interact with protein digestion (Sewell and West, 1965). Eggum (1973)
showed that lactose may improve protein utilization aswell as intestinal pH, microflora
and intestinal vitamin synthesis.Thus, lactose influences protein digestion both directly
and indirectly.
From the foregoing paragraphs it can be concluded that protein digestion is affected by
dietary factors, animal factors and interactions between animal and dietary factors.
In this chapter different aspects of protein digestion have been outlined with special
reference to newly weaned piglets. The digestion of dietary protein is a multi-factorial
processaffected byvariousanimalanddietaryfactors.Digestiveadaptation tothe change
in dietary composition constitutes a major event during the early post-weaning period in
young pigs.
This thesis describes investigations performed with young piglets to elucidate the
physiological adaptations occurring at weaning with respect to the digestion of different
dietary protein sources.
Scope of theThesis
The performance of young piglets (growth, feed intake, feed/gain ratio) depends on
several factors, e.g., age of piglets, environmental conditions (e.g. temperature), social
conditions (social order), feeding conditions (amount of feed, composition of feed) and
21
Chapter I
stress factors.
An important aspect of the feed is the protein source: especially young piglets respond
differently to different protein sources. Proteins of animal origin are dealt with more
easilythanvegetable proteins (WilsonandLeibholz, 1981a,b,c).Thisdifference inprotein
digestion diminisheswith advancing age ofthe animals (Combsetal, 1963;Van de Kerk,
1982).
The research described in this thesis was undertaken to study the
development of the protein digestive capacity of young piglets during the
post-weaning period.
From the foregoing paragraphs it can be derived that the proximal digestive tract
(stomach and duodenum + pancreas) isofmajor importance for the digestion of dietary
protein. Digestive disorders resulting from poor protein digestion may be exhibited
further down the gut, e.g., bacterial proliferation due to excessive amounts of substrate
at the terminal ileum or diarrhoea at the faecal level. However, these (sub)clinical signs
will be preceded by digestive disturbances occurring at the upper digestive tract.
Therefore, it is postulated that the proximal digestive tract organs (stomach and
pancreas) play a key role during the post-weaning adaptation period to a new dietary
(protein) regime. The process of adaptation of the stomach and pancreas with respect
to enzyme secretion and pH regulation will determine the development of (dietary
protein-related) digestive disorders often observed duringthe earlypost-weaning period.
Extending our knowledge on the digestive adaptation of newly weaned piglets may
provide nutritional tools to improve post-weaning pig performance.
To investigate the role of the stomach and the pancreas in the development of postweaningprotein digestivecapacityofyoungpiglets,thefollowing aspectshavebeen taken
into account:
•
•
•
•
Endogenous nitrogen losses at the faecal and ileal level have been determined in
young piglets fed different protein sources to assess the causes of differences in
apparent nitrogen digestibility between protein sources (Chapter II).
Literature data on the role of the pancreas and its adaptation to various animal
and feed characteristics have been collected (Chapter III).
Some analytical aspects of the determination of pancreatic trypsin and
chymotrypsin activity have been investigated (Chapter III and IV).
The development of pancreatic trypsin and chymotrypsin activity in pancreatic
tissue and small intestinal digesta of newly weaned piglets fed different dietary
protein sources has been studied (Chapter V and VI).
22
Chapter I
Gastric protein breakdown and digestive tract pH related to the development of
protein digestive capacity have been examined (Chapter V and VI).
Jejunal wall morphology has been studied with respect tovillus length and crypt
depth (Chapter VI).
Pancreatic secretion has been measured in response to intravenous and
intraduodenal stimuli (Chapter VII).
Interrelationships between gastric protein breakdown, gastrointestinal pH,
pancreatic enzyme activities and nitrogen digestibility have been described
(Chapter VIII).
The role of gastric function, pH, pancreatic enzymes and feed characteristics in
post-weaning piglet performance has been discussed (Chapter VIII).
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ChapterI
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Physiology of Intestinal Digestion and Absorption. Chapter 8 in: Protein
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Chapter I
Hampson, D.J., Kidder, D.E., 1986.
Influence of creep feeding and weaning on brush border enzyme activities in the
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Untersuchungen zuVerdauungsvorgangen beiAbsetzferkeln inAbhangigkeit von
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Leibholz, J., 1981.
Digestion in the pigbetween 7and 35days of age.6.The digestion of hydrolyzed
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The Feeding ofAnimals.Section Vin:AnimalNutrition and Veterinary Dietetics.
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Chapter I
Lofstedt, M., 1986.
Clinical and Physiological Effects of Weaning in Pigs with Special Reference to
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An evaluation of skim milk powders subjected to different heating conditions
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Chapter I
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Ernahrungsphysiologische Untersuchungen an Absetzferkeln. Thesis.
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Some effects of lactose on protein utilization in the baby pig./.Anim. Sci. 24:239241
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Chapter I
Stanton, H.C., Mueller, R.L., 1976
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Wood, W.B., Wilson, J.H., Benbow, R.M., Hood, L.E. (editors), 1974.
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Equal levels of soybean and milk proteins in diets for artificially-reared neonatal
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28
Chapter II
ENDOGENOUS N LOSSES ATTHE TERMINAL ILEUM OFYOUNG PIGLETS
FED DIETS BASED ON FOUR DIFFERENT PROTEIN SOURCES
Caroline A. Makkink1, Thomas Heinz2,3, Wolfgang B. Souffrant2
Martin W.A. Verstegen1
1
2
Agricultural University, Department of Animal Nutrition,
Haagsteeg 4,6708PMWageningen, The Netherlands
Research Institute for the Biologyof Farm Animals,Department of Nutritional Physiology,
"Oskar Kellner",Justusvon Liebigweg 2,2500Rostock, Germany
3
present address:Serumwerk BernburgAG,
Hallesche LandstraCe 105b,D0-4350 Bernburg, Germany
Submitted to British Journal of Nutrition
29
Chapter II
Summary
Fourteen piglets, initially weighing 8 kg, were each fitted with a PVTC (Post Valvular T
Caecum) cannula andwithtwocatheters,one inajugularveinand one in a carotid artery. Each
piglet received one of four experimental diets containing either skimmilk powder (SMP),
soybean meal (SBM),soyprotein isolate (SI) or fish meal (FM) as the sole protein source. The
diets were given in equal amounts at 12 h intervals. 16N-L-Leucine was intravenously infused
continuously at a rate of ± 40mg 15N-L-Leucine per kgbody weight per day.Apparent faecal
nitrogen (N) digestibilities were 94.0,84.0,90.8 and 89.1 % for the SMP,the SBM, the SI and
the FM diet, respectively. Daily endogenous faecal N losseswere 315,963,715 and 697mg for
dietsSMP,SBM,SI and FM,respectively.True faecal Ndigestibilitieswere 97.3,93.5,98.1and
96.4 % for diets SMP,SBM, SIand FM, respectively. Apparent ileal nitrogen (N) digestibilities
were 84.4, 76.5,78.4 and 73.0 % for the SMP, the SBM, the SI and the FM diet, respectively.
Daily endogenous ileal Nlosseswere 786,1422, 1970and 1558mgfor diets SMP,SBM,SI and
FM, respectively. True ileal N digestibilities were 92.7, 90.6, 98.4 and 89.3 % for diets SMP,
SBM,SI and FM,respectively. Itwasconcluded that true N digestibilitieswere high for all four
protein sources,both at the ileal and at the faecal level. Differences in apparent N digestibility
were mainly caused by differences in endogenous N secretion.
Introduction
Apparently digested nitrogen at the terminal ileum is considered to be a good measure for
nutritionalvalue of protein inthe diet.The apparently undigested part,however, can arise from
either the feed protein or from endogenous protein (secreted enzymes, mucus, sloughed off
mucosal cells).To distinguish between endogenous and exogenous nitrogen, different methods
have been proposed (Souffrant, 1991). Until recently, protein free diets and regression
techniques have been used to measure endogenous nitrogen losses and thus calculate 'true'
compared toapparent digestibilities (Souffrant, 1991).However, protein free diets and different
protein sources may itself lead to different levels of endogenous secretion (Huisman, 1990, De
Lange et al, 1989). To obtain results on endogenous secretion in normally fed pigs, the feed
nitrogen or nitrogen in the animal body can be labelled with a stable isotope in order to
distinguish between endogenous and exogenous nitrogen. From that true nitrogen digestibility
(also referred to as 'real' nitrogen digestibility) can be calculated (Souffrant, 1991).
Recently, the 15N infusion method for labelling the animal body nitrogen has been introduced
and evaluated (Souffrant et al, 1981,De Lange et al, 1990). With this technique a distinction
30
Chapter II
can be made between endogenous and exogenous nitrogen at the faecal or ileal level.
Endogenous nitrogen originates from different sources:saliva,gastric and pancreaticjuice,bile,
gut wall secretions, sloughed off gut wall cells and bacteria. The precursor pools for all these
endogenous nitrogen sources cannot be analyzed separately. Therefore, itisimportant to have
an indicator which reflects the endogenous precursor pool. Urine is not suitable as such,
because the amount and composition of nitrogen containing substances inurine depends on the
utilization oforallyingested nitrogen bytheanimal.Wholeblood and plasma contain alsolarger
molecules(proteins) whichwillnotbe usedfor theproduction of compounds tobe secreted into
the lumen. The TCA-soluble fraction of the blood plasma is considered to be the precursor
(amino acids,small peptides, urea) pool for synthesis of endogenous nitrogen. The secretion of
nitrogenous compounds originates thus from both intermediary protein conversion and from
absorbed nutrients (Herrmann etal, 1986,Souffrant etal., 1981,1986,1991). The endogenous
nitrogen indigesta or faeces can be calculated through isotope dilution and hence true nitrogen
digestibilities can be calculated. Since secreted nitrogenous compounds can also be formed in
the enterocytes directly from absorbed resources, the endogenous nitrogen losses as measured
usingtheTCA-soluble plasmaastheprecursor poolwillgenerallybesomewhat underestimated.
The technique assumescomparable absorption oflabelled and unlabelled aminoacids.Also,the
extent of labelling of the endogenous nitrogen secretion should not change significantly during
the course of the experiment. However, because of the continuous (albeit slow) rise in body
protein labelling due to recycling of labelled amino acids,calculated endogenous Nvalues tend
to be underestimated.
The measurement of endogenous N losses can give information on the causes of low apparent
digestibilities (either low digestibility of the feedstuff or high endogenous secretion by the
animal).
Especially with young animals, low apparent (nitrogen) digestibilities are sometimes found at
theileal and the faecal level,especiallywhenvegetable protein (e.g.,soyprotein) isfed in stead
of protein of animal origin (e.g., milk proteins).
Not much isknown about the causes of low(apparent) digestibilities of different dietary protein
sources in young piglets. Digestibility of soy protein is lower for piglets than for older pigs,
digestibility of milk protein is high for pigs of all ages (Wilson & Leibholz, 1981a,b,c).
For the development of feeds for newly weaned piglets it is important to know endogenous
nitrogen lossesandapparent nitrogendigestibilityafter weaning.Anexperimentwasundertaken
to compare endogenous nitrogen losses in young piglets fed diets based on different protein
sources.
The method of infusion of 16N-L-leucine was used to assess the endogenous N losses in the
faeces and at the distal ileum in young piglets. The 15N label will also appear in other (though
31
ChapterII
not all) amino acids due to transamination.
Due to different levelsof transamination, it isnot feasible to measure the separate endogenous
amino acid losses.
Materialsand Methods
Animals, diets and experimental scheme
Fourteen castrated male piglets (Great York, initial live weight 7.5 - 8.5 kg) were used to
measure apparent and true ileal nitrogen digestibilities of four different diets. Piglets did not
receive any creep feed during the sucklingperiod. Pigletswere weaned between 3and 4weeks
of age and housed individually on mesh floors, tethered to prevent cannula and catheter
damage.
Piglets were fed twice daily at 8.00 h and 20.00 h. Two piglets were fed the diet based on
skimmilk powder (SMP), three piglets were fed the diet based on soybean meal (SBM), six
piglets (four experimental and twospare piglets)were fed the diet based on soyisolate (SI) and
three piglets were fed the diet based on fish meal (FM). The diets were fed at a level of 380
g per day throughout the experiment (approx. 4% of live weight per day).
Composition of the protein sources and diets isgiven inTable 1and 2.The dietswere balanced
with regard to net energy, crude protein, crude fat, crude fibre and essential amino acids.
Chromium oxide at a level of 0.1 % was included as a marker for digestibility calculation.
After a 6 day 'cage adaptation' period and an overnight fasting period, the piglets were
anaesthetized through inhalation anaesthesia (N 2 0, 0 2 , halothane) and fitted with a 'Post
Valvular T Caecum' cannula (Van Leeuwen et al, 1991) at an age of 4 to 5 weeks
(approximately 8 kg live weight). Immediately before surgery, lidocaine with epinephrine was
administered, subcutaneously and intramuscularly inthe area of theincision.The flank areawas
shaved and disinfected with a general disinfectant. An incisionwasmade inthe right abdominal
wall in the entera-paralumbar area and some distance above the mammary tissue. The caecal
apex and the terminal portion of the ileum were located, exteriorized and the caecum was
removed.The ileum and the large intestine at the siteof the removed caecum was exteriorized.
The cannula barrel (internal diameter 19mm, external diameter 24 mm) was placed through
the opening which was made not larger than necessary to aid in securing and positioning the
cannula barrel. Thus, surgery consisted of a caecectomy in which an opening for the cannula
appeared directly opposite the ileocaecal valve (Van Leeuwen et al, 1991).
32
Chapter II
Composition of the protein sources, nutrients, amino acids and trypsin inhibitor activity
Table 1.
protein source:
dry matter (DM),%
ash (% of DM)
crude protein (CP, %of DM)
ether extract (% of DM)
crude fibre (% of DM)
N-free extracts (% of DM)
SMP
skimmilk
powder
96.2
8.3
35.4
0.5
0.0
55.8
Lysine (% of CP)
Threonine (% of CP)
Methionine (% of CP)
Cystine (% of CP)
Tryptophan (% of CP)
Trypsin inhibitor
(mg inhibited trypsin per g
Figure 1.
SBM
soybean
meal
87.5
7.1
52.6
1.7
7.1
31.5
SI
soy protein
isolate
95.2
4.1
89.5
3.6
1.3
1.5
FM
fish
meal
94.4
13.8
75.8
10.3
0.1
0.0
8.24
4.23
2.70
1.00
1.30
5.84
3.72
1.39
1.61
1.22
6.01
3.66
1.21
1.12
1.04
7.52
4.04
3.01
1.02
0.96
_
3.64
6.27
_
product)
Post Valvular T Caecum cannula in place (Van Leeuwen et al.,
A. directly after opening the cannula,
B. about 15minutes after opening of the cannula
(ileocaecal valve protrudes into the cannula)
1.ileocaecal valve
2. caecum
3. colon
33
1991).
4. PVTC cannula
Chapter II
Table 2.
Diet composition
diet:
ingredient (%of the diet}
skimmilk powder (35.3 %CP)
soybean meal (48 %CP)
soy isolate
fish meal
maize starch
dextrose
sunflower/soy oil
cellulose
vit/min premix
ground limestone
mono Ca phosphate
salt
KHCO s
NaHC0 3
L-Lysin-Hcl
DL-Methionine
L-Threonine
L-Tryptophan
Cr 2 O s
SMP
skimmilk
powder
45.50
29.60
15.00
2.00
5.00
1.00
0.80
0.50
0.10
0.30
0.10
0.1
analyzed comDOsition(%)
dry matter (DM)
91.0
nutrients (% in drv matter)
crude protein (CP)
16.9
crude fibre
4.1
LYS
1.39
MET
0.56
CYS
0.17
THR
0.71
TRP
0.22
metabolizable energy (MJ/kg DM) 16.63
net energy (MJ/kg DM)
11.64
SBM
soybean
meal
SI
soy protein
isolate
FM
fish
meal
-
-
-
34.40
39.84
15.00
2.00
2.85
1.00
1.35
2.10
0.50
0.40
0.16
0.20
0.10
18.20
52.79
15.00
2.00
5.00
1.00
1.40
2.20
0.50
1.50
0.10
0.17
0.04
-
-
0.1
0.1
22.15
52.91
15.00
0.50
5.00
1.00
0.75
0.60
0.10
1.00
0.80
0.03
0.05
0.01
0.1
89.8
90.7
89.6
18.4
5.7
1.21
0.48
0.30
0.79
0.22
16.51
11.56
17.6
4.2
1.14
0.40
0.20
0.69
0.18
17.00
11.90
18.4
4.4
1.39
0.59
0.19
0.81
0.19
17.30
12.11
This cannula allows nearly quantitative collection of ileal chyme (Kohler etal., 1992). Figure 1
shows the location of the cannula. After surgery, a 7 days recovery period was allowed before
blood vessel catheterization. Catheters were placed in the externaljugular vein (for continuous
15
N infusion) and in the carotid artery (for blood sample collection). After catheterization,
another 4 days recovery period followed before 15N infusion started.
34
ChapterII
The continuous intravenous 15N-L-Leucine infusion was performed at a rate of approximately
40 mg 15N-L-Leucine (95 % 15N enrichment) per kg body weight per day (Perfusoi^-SecuraDauerinfusionsgerat, Fa. B. Braun, D-3508 Melsungen). Two additional piglets on the SI diet
were excluded from 15N infusion.
Sample collection and analysis
From the start of 15Ninfusion quantitative faeces and urine collectionsweremade daily.During
the first 6 days of infusion the PVTC cannula was closed. Apparent and true faecal N
digestibilities were determined from the ingested feed and the excreted faeces during these 6
days. Faeces were collected in plastic bags attached to the animals by means of a colostomy
system as used inhumane medicine (CombihesiveR-system,Squibb BV,NL 2285VL Rijswijk).
Quantitative urine collections were made and twice daily aliquots were taken and frozen for
nitrogen determination. At day 7, 9 and 11 after the start of 15N infusion, ileal chyme was
collected for 24hours per day.Digesta were collected, weighed and frozen hourly, pooled per
animal per day.Digesta and faeces were freezedried and ground (1mm) before analysis. Feed
samples were also ground before analysis. Apparent ileal and faecal N digestibilities were
determined from the concentration of marker (Cr 2 O s ) in feed and in digesta or faeces. Cr 2 O s
was analyzed in feed, digesta and faeces through flame atom absorption spectrophotometry
(Perkin-Elmer 300).
Trypsin inhibitor contents were analyzed according to Van Oort et al. (1989).
Bloodsamplesweretaken twicedailyfrom the carotid catheter duringfeeding at8.00 and20.00
hours. After centrifugation (2500 rpm, 10 minutes), the supernatant was collected and blood
plasma protein was precipitated using 20% trichloroacetic acid (TCA). Total N was analyzed
in supernatant, precipitate, digesta, urine and faeces according to the Kjeldahl method. After
Kjeldahl-N-analysis, the amount of 15Nwasmeasured in the NH4Cl-solutions using an emission
spectrometer (Isonitromat 5201 or NOI-6, Fa. Statron, Furstenwalde, Germany).
The corrections for ileal and faecal endogenous nitrogen were made from the 1BN enrichment
excess in TCA soluble fraction of blood plasma and in digesta or faeces during the days of 16N
infusion.The contribution of endogenous to total N in ileal chyme or faeces can be calculated
from the ratio of 15N enrichment excess in ileal chyme or faeces and in the blood TCA soluble
fraction, assuming that the 15N excess in the endogenous N and in the blood TCA soluble
fraction issimilar.The calculations are carried out according to Souffrant etal.(1986) using the
following formula:
35
Chapter II
N,exc/f
N.n = N tot X
N,exbl
N tot = total N in chyme or faeces (g/day)
N en = endogenous N in chyme or faeces (g/day)
Nexc/f = U N excess in chyme or faeces
N, bl = 15N excess in the TCA soluble fraction
of the blood
True nitrogen digestibilitieswere calculated from the apparent digestibilities bysubtracting the
endogenous nitrogen from the total nitrogen recovered from faeces or ileal chyme.
Statistical analysis was done by one-way analysis of variance and Tukey multiple comparisons
(a=0.05) were used to compare means of experimental groups (Snedecor, 1956; Rasch et al,
1978, SAS, 1985).
Results
In Figure 2, 15N enrichment excess in digesta, faeces, urine and TCA-soluble blood plasma
during 15N-L-leucine infusion is shown. After 6 days (=144 hours) of infusion, the percentage
enrichment levels off indicating sufficient labelling of animal body protein.
Results on faecal digestibilities and losses are presented in Table 3.
o.ao
15N enrichment excess (%)
0.20
TCA aol pi
o.io
0.00
100
200
300
time after start infusion (hours)
Figure 2.
15
Nenrichmentexcess(%)inurine,faeces,ilealchymeandTCA-solubleplasma,soybean
mealdiet (means of threeanimals).
36
Chapter II
Table 3.
16
Resultsof N infusion experiment, digestibility andendogenous losses,faecal data,
diet
SMP
SBM
SI
FM
rootMSE
number of piglets
apparent faecal
N digestibility (%)
2
3
5*
3
94.0a
84.0b
90.7a
89.1 a
number of piglets
endogenous faecal N
mg/day
endogenous faecal CP
g/lOOg DM intake
endogenous faecal N
g/100 g N intake
endogenous faecal CP
mg/kg LW
true faecal
N digestibility (%)
2
3
4
3
315b
963 a
715 ab
697 ab
204.7
0.56b
1.77a
1.30ab
1.28ab
0.372
72ab
2.06
3.4
b
9.6
a
7.4
ab
1.75
174b
561 a
425 ab
404 ab
120.8
97.3 a
93.5 b
98.1 a
96.4a
0.78
Means within a row bearing the same superscript do not differ (Tukey, p>0.05)
different number of piglets for apparent digestibility is caused by inclusion of one of the two
spare piglets without N-infusion on the Si-diet.
Apparent faecal N digestibility was highest in piglets fed the SMP diet (94.0%) and lowest for
the SBM diet (84.0%). Apparent faecal N digestibilities for the SI and FM diets were
intermediate (90.8and 89.1%,respectively).Endogenous Nlossesatthefaecal levelwerelowest
for the SMP diet (315 mg/d) and highest for the SBM diet (963 mg/d). Endogenous N losses
on the SI and FM diets were intermediate (715 and 697 mg/d, respectively). True faecal N
digestibility was highest for the SI and SMP diets (98.0and 97.3%, respectively) and lowest for
the SBM diet (93.5%).True faecal N digestibility was lowest on the SBM diet (93.5%). True
faecal N digestibility on the FM diet was intermediate (96.4%). True faecal N digestibilities
were high (> 90%) on all diets.
Data on ileal digestibilities and N losses are given in Table 4.
Apparent ileal N digestibility was highest with the SMP diet (84.4 compared to 78.4, 76.5 and
73.0 % for the SI,the SBM and the FM diet respectively). Endogenous Nlossesat the terminal
ileum were about twice as high with the SBM diet and the FM diet compared to the SMP diet,
resulting in almost similar true ileal N digestibilities for the three diets (92.7,90.6 and 89.3 %
with the SMP, the SBM and the FM diet, respectively).
37
Chapter II
16
Table4.
Resultsof N infusion experiment, digestibility and endogenous losses,ilealdata,
diet
SMP
SBM
SI
FM
rootMSE
number of piglets
apparent ileal
N digestibility (%)
2
3
6*
2"
84.4a
76.5b
78.4 ab
73.0b
number of piglets
endogenous ileal N
mg/day
endogenous ileal CP
g/100 g DM intake
endogenous ileal N
g/100 g N intake
true ileal
N digestibility (%)
2
3
4
2**
786b
1422ab
1970"
1558ab
359.3
1.41b
2.6l a b
3.56a
2.86 ab
0.641
8.3b
14.1 ab
20.4a
16.2ab
3.69
t
92.7
b
90.6
b
98.4
a
89.3
b
2.67
1.29
Meanswithin arow bearing thesamesuperscript donot differ (Tukey, p>0.05)
different number of piglets for apparent digestibility is caused by inclusion of two spare piglets
without N-infusion.
one piglet on FM diet was excluded due to aberrant values on nitrogen content in ileal digesta.
With theSIdiet endogenous losseswere about 2.5timeshigher compared to the SMPdiet.The
true ileal N digestibility for the SI diet was 98.4 %.
Apparent N digestibilities (faecal and ileal) did not differ between SBM and SI diet (p>0.05,
Table 3 and 4), however, true digestibilities (faecal and ileal) did differ (p<0.05,Tables 3 and
4). The SMP caused higher apparent N digestibilities (faecal and ileal) than the other protein
sources.
In general, differences in true N digestibilities between protein sources were smaller than
differences in apparent N digestibilities.
With the soy-based diets (SBM and SI) standard deviations of parameters were generally larger
than withthe animal protein diets (FM and SMP).Thisindicates that large differences between
individual piglets were noticed when soy-based diets were fed.
The soy-based dietscaused the highest endogenous nitrogen losses (SIat the terminal ileal level
and SBM at the faecal level).The true ileal digestibility of the SIprotein washigher than of the
other protein sources.
Nitrogen balance data and organ weights are given in Table 5.
38
Chapter II
Table 5.
Organ weights and Nbalance data, from day 1till day6.
number of animals(n)
live weight(kg)
pancreatic weight (g)
small intestinal weight (g)
N intake (mg/day)
N urine (mg/day)
N urine as%of N intake
N faeces (mg/day)
N faeces as%of N intake
N balance (mg/day)
N balance as%of N intake
SMP
SBM
SI
FM
rootMSE
2
11.3a
19.3 ab
317a
9438c
1935ab
20.5 ab
568b
6.0b
6936s
73.5a
3
10.7a
22.1 ab
375a
10048"
2014b
20.0b
1611"
16.0a
6423 ab
63.9 ab
5"
10.4a
23.0a
401 a
9646b
3409a
35.4a
1040ab
10.8 ab
5198b
53.9b
3
10.8a
15.9b
355a
9626b
2171 ab
22.6 ab
1048ab
10.9ab
6407 ab
66.6 ab
0.52
2.53
75.4
27.0
576.7
6.07
304.6
3.12
582.4
5.91
Means within arowbearing the same superscript donotdiffer (Tukey, p>0.05)
including onespare piglet without 15N-infusion.
Table 6.
Results ofcomparisons ofmeans (T-tests) for ileal vsfaecal andtrue vsapparent datafor
each protein source, diff. =difference between faecal and ileal or between apparentand
true. When p<0.05 'diff.' issignificantly different from 0.
diet:
SMP
diff. (p)
SBM
diff. (p)
SI
diff. (p)
FM
diff. (p)
App. N dig.:
End. Nloss
(mg/day):
Endogenous CP
(g/lOOg DMint.):
Endogenous N
(g/100gNint.):
End. CP(mg/kgLW):
True N dig.:
9.65 (0.0099)
7.50 (0.0053)
12.75 (0.0060)
15.90 (-)
-471.(0.0041)
-459(0.0158)
-1256(0.0297)
-880(0.1065)
-0.85 (0.0038)
-0.84(0.0156)
-2.27 (0.0284)
-1.62(0.1035)
-4.95 (0.0064)
-260.5 (0.0041)
4.60 (0.0277)
-4.57 (0.0147)
-268.0 (0.0184)
2.90(0.0151)
-13.00(0.0168)
-733.6 (0.0338)
-0.33 (0.7471)
-9.15(0.1001)
-503.7 (0.1246)
6.75 (0.1437)
APPARENT-TRUE
SMP
diff. (p)
SBM
diff. (p)
SI
diff. (p)
FM
diff. (p)
ileal N dig.:
faecal N dig.:
-8.35 (0.0267)
-3.30 (0.0768)
-14.17(0.0221)
-9.57 (0.0356)
-20.48 (0.0031)
-7.40 (0.0053)
-14.17(0.0221)
-7.10(0.0448)
FAECAL-ILEAL
39
Chapter II
Nodifferences inliveweightsbetween dietswerenoticed (p>0.05).Pancreaticweightsdid differ
between diet SI and diet FM (p<0.05). Small intestinal weights did not differ between diets
(p>0.05).
Nitrogen intake differed between diets, due to different crude protein contents in the diets.
Urinary N excretion was highest with the SI diet and lowest with the SMP diet. Faecal N
excretion was highest with the SBM diet and lowest with the SMP diet. The difference in N
balance between SMP and SI was significant (p<0.05).
InTable 6comparisons are givenbetween faecal and ileal data andbetween apparent and true
digestibilities.
At the ileal level and at the faecal level, true N digestibility was higher than apparent N
digestibility with all protein sources (Table 7). The ranking for apparent ileal N digestibility
(SMP-SI-SBM-FM) was not similar to the ranking for true ileal N digestibility (SI-SMP-SBMFM).
Discussion
Endogenous nitrogen infaeces and ilealchyme asmeasured usingthe continuous 1SN-L-Ieucine
infusion technique can be underestimated for two reasons: Firstly, endogenous protein is
synthetized not only from free amino acids in the TCA-soluble fraction of the plasma, but also
directly from absorbed amino acids in the enterocytes of the intestinal wall. Secondly, the
labelling ofbodyprotein willcontinue torise (slowly) duringcontinuous infusion of 15N-leucine,
due to recycling of labelled amino acids. This will also result in an underestimation of the
amount of endogenous nitrogen (De Lange et al, 1990).
Wilson & Leibholz (1981c) studied apparent and true N digestibilities of diets containing milk
or soybean protein with piglets (28days of age) using a nitrogen free diet.For the milk protein
diet they found apparent and true ileal N digestibilities of 86 and 92 % respectively, which
closely resembles our values of 84 and 93 % respectively. Apparent nitrogen digestibility was
expected to be higher for the milk protein diet because the newly weaned pig is supposed to
be adapted to this protein source. The relatively low digestibility could be due to differences
between cow's milk protein and sow's milk protein.
Wilson & Leibholz (1981c) reported apparent and true ileal N digestibilities for the soybean
meal diet of 51 and 62 % respectively. In our study we found apparent and true ileal N
digestibilities for the SBM diet to be 77and 91% respectively. This difference could be due to
age of piglets (in our study 35 - 50 days), to nitrogen content of the diet (Wilson & Leibholz
40
Chapter II
(1981c) used diets containing ± 27 % of crude protein), to quality of soybean meal or to an
interaction between age and diet composition.Thislatter option hasbeen supported byWilson
& Leibholz (1981a) who studied performance of piglets given milk and soybean proteins at
different ages. They found an interaction between protein source in the feed and age of the
piglets: the performance of piglets fed milk protein did not change between 7 and 35 days of
age. Performance of piglets fed soybean protein, however, did increase with increasing age.
Wilson & Leibholz (1981c) studied endogenous N flow at different sites of the gastrointestinal
tract of piglets (35 days of age) fed a protein free diet. They reported an endogenous N flow
at the terminal ileum of 0.82 gN per day. In our studywe found N flows of 0.79 and 1.42 gN
per day for piglets fed the SMP diet and the SBM diet respectively. These data indicate that
endogenous losses are minimal when milk proteins are fed to young piglets.
Endogenous N losses measured with animals fed protein free diets possibly do not reflect the
normal, physiological situation when protein containing diets are fed. The fact that different
protein sources in the feed induce different levels of endogenous N secretion stress this aspect.
Since the amount of dietary nitrogen could also affect the endogenous nitrogen losses, the
regression method also is not very accurate in determining endogenous nitrogen losses in
animals fed nitrogen containing diets.
Leibholz (1982) studied the endogenous nitrogen secretion inyoung pigs (4weeks of age).She
used the regression method to assess endogenous N losses with a feed based on milk protein.
Bymeans of regression analysis she calculated endogenous N flow at the terminal ileum to be
3.44 gN per kg dry matter intake, which amounts to 2.2 g endogenous ileal crude protein per
100gof drymatter intake. In our studywecalculated 1.41 gendogenous ileal crude protein per
100g of dry matter intake for the SMP diet.
The weight of the pancreas isinagreement with data from Le Guen etal.(1991)obtained from
piglets of 10 to 15 kg live weight fed diets based on casein and fishmeal or pea proteins. The
difference in pancreatic weight between SI and FM fed piglets is difficult to explain: it is
assumed (Huisman, 1990), that piglets do not show pancreas hypertrophy when fed trypsin
inhibitor containing feedstuffs. The reason for the low pancreatic weight of FM fed piglets is
unclear.
The low Nbalance of SI fed piglets is caused byboth urinary and faecal N excretion. The high
faecal N excretion is caused by both endogenous and exogenous nitrogen. With young piglets
(age 35 to 56 days), Newport and Keal (1983) found no effect of dietary protein source (soya
bean meal versus combinations of fish meal, skimmilk powder and soybean meal) on nitrogen
retention with comparable nitrogen intakes.With diets containing fish meal, soybean meal and
skimmilk powder, Zhang etal.(1986) found a Nretention of7.1 g/daywhen 10.9gram ofNwas
41
Chapter II
ingested daily by piglets aged 31 days on average.
From our data we concluded that the differences in apparent ileal N digestibility between the
SMP, SBM and FM diet are not caused by differences in the (true) digestibility of the protein
sources.From our experiment we also concluded, that true ileal Ndigestibility of soybean meal
isnot much different from true ileal N digestibility of skimmilk powder. Therefore, differences
between these protein sources with regard to apparent ileal N digestibility and performance of
piglets(growth,feed conversion ratio)mustbe causedbyan increase inendogenousN secretion
with pigletsfed soybean meal.The soyprotein itself ishighlydigestible (90.6 % at the ileallevel
and 93.5%at the faecal level),but it either stimulates nitrogen secretion bythe exocrine glands
of the digestive tract or it causes excessive loss of gut wall cells by sloughing off, resulting in
endogenous N losses. These endogenous N losses may lead to decreased performance (feed
intake, growth) of piglets fed non-milk protein sources after weaning.
In terms of endogenous losses, the expenses for the digestion of vegetable protein are higher
than for the digestion of milk protein.
References
Guen, M.P. le, Huisman, J., Makkink, C.A., 1991.
Effect of peas and pea isolates on protease activities in pancreatic tissue of piglets.
ProceedingsoftheVthInternationalSymposiumonDigestivePhysiologyinPigs, Wageningen
(Doorwerth), Netherlands, (EAAP PublicationNo 54, 1991),editedbyM.WA. Verstegen,
J.Huisman and LA. den Hartog PUDOC, Wageningen, 1991,pp 207-210
Herrmann, U., Krawielitzki, K., Smulikowska, S., Schadereit, R., 1986.
Zur Bestimmung des endogenen Stickstoffs im Kot mit Hilfe der Isotopentechnik.
Archives ofAnimal Nutrition 36:857-865
Huisman, J., 1990.
Antinutritional effects of legume seeds in piglets, rats and chickens. Thesis, Agricultural
University, Wageningen,TheNetherlands,149pp.
Kohler, T., Mosenthin, R., Verstegen, M.W.A., Huisman, J., Hartog, L.A. den, Ahrens, F.,
1992.
Effect of ileo-rectal anastomosis and post-valve T-caecum cannulation on growing pigs.
1.Growth performance, N-balance and intestinal adaptation.BritishJournalofNutrition
68:293-303
42
ChapterII
Lange, C.F.M. de, Sauer, W.C., Mosenthin, R., Souffrant, W.B., 1989.
The effect of feeding different protein-free diets on the recovery and amino acid
composition of endogenous protein collected from the distal ileum and feces in pigs.
Journal ofAnimal Science67(3):746-754
Lange, C.F.M. de, Souffrant, W.B., Sauer, W.C., 1990.
Real ileal protein and amino acid digestibilities in feedstuffs for growing pigs as
determined withthe 15N-isotopedilution technique.JournalofAnimal Science68(2):409418
Leeuwen, P.van, Kleef, D.J.van, Kempen, G.J.M.van, Huisman, J., Verstegen, M.W.A.,1991.
The post valve T-caecum cannulation technique in pigs applicated to determine the
digestibility of amino acids in maize, groundnut and sunflower meal.Journalof Animal
Physiology andAnimal Nutrition 65:183-193
Leibholz, J., 1982.
The flow of endogenous nitrogen in the digestive tract of young pigs.British Journalof
Nutrition48:509-517
Newport, M.J., Keal, H.D., 1983.
Effect of protein source on performance and nitrogen metabolism of pigsweaned at 21
days of age.Animal Production 37:395-400
Oort, M.G. van, Hamer, R.J., Slager, E.A., 1989.
The trypsin inhibitor assay: improvement of an existing method.In: RecentAdvances of
ResearchinAntinutritional FactorsinLegume Seeds. Eds.J. Huisman, A.F.B. vanderPoel
& I.E. Liener. Pudoc, Wageningen,TheNetherlands.
Rasch, D., Herrendorfer, G., Bock, J., Busch, K., 1978.
Verfahrensbibliothek Versuchsplanung und -auswertung. VEB Deutscher
Landwirtschaftsverlag, Berlin.
SAS Institute Inc., 1985.
SAS User's Guide: Statistics. Version 5 Edition. Cary, NC: SAS Institute Inc., 1290pp.
Snedecor, G.W., 1956.
Statistical Methods. 5th edition;Iowa State College Press, Ames, Iowa.
Souffrant, W.B.,1991.
Endogenous losses during digestion in pigs. Proceedings of the Vth International
Symposium on DigestivePhysiology in Pigs, Wageningen (Doorwerth), The Netherlands,
(EAAP Publication No 54, 1991), edited byM.W^A. Verstegen, J.Huisman and L~A. den
Hartog,PUDOC, Wageningen, 1991,pp147-166
43
ChapterII
Souffrant, W.B., Kohler, R., Matkowitz, R., Gebhardt, G., Schmandke, H., 1981.
Ernahrungs-physiologische Untersuchungen an Schweinen zur Beurteilung von
modifizierten Proteinen. 2.Bestimmungdes endogenen N im Dunndarminhalt mit Hilfe
der 15N-Tracertechnik.Archivfur Tieremahrung31:675-683
Souffrant, W.B.,Darcy-Vrillon, B., Corring, T., Laplace, J.P., Kohler, R., Gebhardt, G.,
Rerat, A., 1986.
Recyclingof endogenous nitrogen inthe pig:Preliminary results ofa collaborative study.
Archives ofAnimal Nutrition 36:269-274
Wilson, R.H., Leibholz, J., 1981a.
Digestion inthe pigbetween 7and 35daysofage. 1.The performance ofpigsgivenmilk
and soyabean proteins.British Journalof Nutrition 45:301-319
Wilson, R.H., Leibholz, J., 19816.
Digestion in the pig between 7 and 35 days of age. 2.The digestion of dry matter and
the pH of digesta in pigs given milk and soyabean proteins.British Journal ofNutrition
45:321-336
Wilson, R.H., Leibholz, J., 1981c.
Digestion in the pig between 7 and 35 days of age. 3.The digestion of nitrogen in pigs
given milk and soyabean protein. British Journalof Nutrition 45:337-346
Wiinsche, J., Herrmann, U., Meinl, M., Hennig, U., Kreienbring, F., Zwierz, P., 1987.
EinfluB exogener Faktoren auf die prazakale Nahrstoff- und Aminosaurenresorption,
ermittelt an Schweinen mit Ileo-Rektal-Anastomosen. 1. Mitteilung. EinfluB des
Zerkleinungsgrades von Getreide.Archives ofAnimal Nutrition 37:745-764
Zhang, Y., Partridge, I.G., Mitchell, K.G., 1986.
The effect of dietary energy level and protein:energy ratio on nitrogen and energy
balance, performance and carcass composition of pigsweaned at 3weeksof age. Animal
Production 42:389-395
44
Chapter III
PANCREATIC SECRETION IN PIGS
Caroline A. Makkink, Martin W.A. Verstegen
Agricultural University, Department of Animal Nutrition,
Haagsteeg 4,6708PM Wageningen, The Netherlands
Published in: Journal of Animal Physiology and Animal Nutrition 64 (1990) 190-208
Reproduced by permission of Paul Parey Verlag, Hamburg
45
ChapterHI
Summary
The pancreas plays a major role in digestive processes. Pancreatic secretion controls
duodenal pH (buffering capacity) and contains digestive enzymes which adapt to diet
composition (substrate content). The development of pancreatic enzymes with age is
important when designing feeding strategies for early weaned piglets.
This paper reviews some aspects of the measurement of enzyme activities in pancreatic
juice and summarizes the effects of age and diet composition on pancreatic (enzyme)
secretion.
The physiological background of adaptation mechanisms is discussed. Some
recommendations for further research in this field are also included.
Introduction
Designing optimal feeding strategies for different categories of farm animals requires the
understanding of digestive processes.
The development of fistulation and cannulation techniques has facilitated studies on
secretion and digestion in vivo.
In this respect the study of pancreatic secretion is important.
The buffering capacity, protein content and enzyme content of pancreatic juice play an
important role in the digestive process. Digestive disorders, e.g., post weaning diarrhoea,
could be related to pancreas function.
Secretion of pancreatic juice probably adjusts to the pH of digesta leaving the stomach.
Digesta leaves the stomach at a low pH (2 to 4), at which pancreatic enzymes are not
active.Therefore, pancreaticjuice must be capable of rising digesta pH to 7a 8to ensure
optimal enzyme activity.
Buffering capacity (acid binding capacity) of feedstuffs isusually defined as the amount of
acid (HC1) needed to lower pH of a feed-in-water suspension by one pH-unit.
Buffering capacity of pancreatic juice should be defined as the amount ofjuice needed to
raise pH of digesta leaving the stomach to pH 7 or 8.
Enzymes should be secreted in amounts appropriate for the digestion of the ingested feed
components. Therefore, enzyme secretion must adapt to dietary composition.
Furthermore, enzymes are proteins which are secreted into the gut lumen. These enzyme46
ChapterHI
proteins will be partly digested and absorbed. Another part will disappear into the large
intestine without being digested or absorbed. This latter part of enzyme-proteins is lost for
utilization by the pig.
The loss of these endogenous proteins should be minimized to ensure optimal feed
utilization by the animal.
Since the development of pancreatic fistulation techniques (Wass, 1965; Pekas, 1965;
Aumaltre, 1972; Corring et al, 1972; Corring and Jung, 1972; Corring and Saucier, 1972;
Corring, 1974; Corring, 1980; Partridge et al, 1982; Schumann et al, 1983; Zebrowska et
al, 1983; Hee et al, 1985; Zebrowska, 1985; Van Leeuwen, 1986; Ozimek et al, 1986;
Pierzynowski etal, 1988)pancreatic secretion hasbecome an important research field over
the past decades.
Pancreatic juice production (flow rates) has been measured in relation to time of day
(Kvastnitskii, 1951),feeding and frequency of meals (Corringetal, 1972;Hee etal, 1988b)
and diet composition (Zebrowska et al, 1981; Langlois et al, 1987; Hee et al, 1988a;
Imbeah et al, 1988).
Abelloetal.(1987) showed that infed pigs,pancreatic and biliarysecretionshardly affected
intraduodenal pH. In fasted pigs, however, bile and especially pancreatic juice did
contribute highly to the neutralization of gastric acid chyme entering the duodenum.
Direct measurements ofbuffering capacity of bile or pancreaticjuice (titration studies) are
not known from literature.
Pancreatic juice contains the following digestive enzymes and zymogens: amylase, lipase,
colipase, trypsinogen, chymotrypsinogen, pro-carboxypeptidase A and B, pro-elastase,
ribonucleases and desoxy-ribonucleases (Bell Davidson Emslie-Smith, 1972b).
The measurement of enzyme activitiesinpancreaticjuice encounters severalproblems,e.g.,
standardization of methods of analysis, enzyme activity changes in stored pancreatic juice
and interference by microbial contamination of the juice.
Methodsfor Collecting PancreaticJuice
The collection of pancreaticjuice from fistulated animals has several advantages: secretion
can be measured directly in response to several factors, e.g., diet composition. Repeated
collections can be made on the same animal, development of secretion with age can be
measured and the juice can be analyzed for protein content, buffering capacity, enzyme
47
Chapter III
activities etc.
The use of fistulation techniques should be subject to critical evaluation: surgery should be
relativelysimpletoperform and measurements shouldberepresentative for normal,healthy
animals.
Basically,twodifferent techniques arebeingused for fistulation ofthe pancreas inpigs.The
first method, developed by Corring et al. (1972) involves fistulation of the pancreatic duct
(see Figure 1).
Figure 1.
Pancreatic duct fistulation technique (Corring etal.,1972)
Hee et al. (1985) use another surgical procedure: they cannulate an isolated segment of
duodenum ("pouch") which receives the pancreatic duct (see Figure 2).
Sometimes one of these two techniques isused with small modifications (Zebrowska et al,
1983 and Pierzynowski et al, 1988).
Both techniques have their advantages and disadvantages: the pouch technique allows the
animals to be used for pancreatic juice collection during several months to over a year.
Pancreatic duct fistulas remain functional for a few weeks maximum.
48
Chapter III
/
^
cannula
duodenum
Figure 2.
Pancreatic cannulation with thepouch technique (Heeetal.,1985)
Withthepancreaticductfistulation technique "clean"and non activated pancreaticjuicecan
be collected while the pouch technique can cause contamination of the juice by duodenal
pouch contents (mucus, dead cells) as well as activation of the juice by duodenal
enterokinase. The pouch technique involves the cutting of the gut at two sites which could
affect gut motility. Both surgery techniques can cause leakage of pancreatic juice into the
abdomen leading to peritonitis and even death of the animal.
There hasnotyet been adirect comparison between thetwodifferent fistulation techniques
with regard to animal health, secretion rates and juice characteristics.
Methodsfor Assessing Pancreatic Enzyme Activity
The activity of pancreatic enzymes can be measured in pancreatic tissue (slaughtered
animals), in pancreatic juice (fistulated animals), in digesta (slaughtered or cannulated
49
ChapterIII
animals), inblood plasma or serum (Frobish etal, 1971) and infaeces (Nitsan and Liener,
1976).
Only small enzyme-molecules (e.g., lipase) can be found in the blood (cell leaking or
absorption). Small amounts of digestive enzymes (not broken down, not absorbed) can be
found in faeces.
The mode of action of enzymes can be represented as follows:
enzyme +
substrate
-» [enzyme-substrate]c
«-
lex
-»
enzyme +
product(s)
This reaction can be followed spectrophotometrically (absorbance change due to increase
in colored product or decrease in colored substrate) or titrimetrically (when substrate or
product possesses acidic properties).
Enzyme activity is usually expressed as units of activity (/xmol/min):
One unit of activity is the amount of enzyme that forms one jumol product per minute or
breaks down one /nmol substrate per minute under standardized assay conditions (pH,
temperature, buffer composition) (Bergmeyer and Gawehn, 1978).
The measured activity depends on the assay method, the pH and the temperature in the
assay and on the substrate used (Bell Davidson Emslie-Smith, 1972a). To ensure
comparability of test results it is necessary to use standardized units in expressing enzyme
activities.
Enzyme activity can be calculated as units per kg liveweight, per gram of pancreatic tissue
(dry or wet), per ml pancreatic juice, per mg of enzyme (specific activity) or per mg of
protein present in the pancreaticjuice. The enzyme activity can also be expressed as total
activity (per pancreas or per 24 hours).
Using fistulation techniques, enzyme activity can be expressed per mlof pancreaticjuice or
as total activity per 24 hours.
Evaluation of Methods toAnalyze PancreaticEnzyme Activities
Literature data on enzyme activities cannot be compared directly because of the many
different assay procedures that are being used with regard to substrate, temperature, pH.
Further difficulties in interpreting pancreatic enzyme assay results are caused by activation
50
Chapter III
of enzyme precursors (trypsinogen, chymotrypsinogen), the standardization of the time
interval between last feed intake and slaughter (when tissue samples are obtained) or
pancreatic juice sampling and the storage of the samples until analysis.
A number of factors can affect the stability of enzymes during storage: extreme pH-values,
heat, freezing and thawing, micro-organisms etc. (Bergmeyer and Gawehn, 1978).
Freezing and thawing of samples has according to Lindemann etal.(1986) no influence on
the activity of trypsin, chymotrypsin, amylase or the gastric proteases.
Gorrill and Friend (1970) kept their samples of pancreatic tissue and ileal digesta at -10°C
after adding glycerol to the digesta samples (1:1) and found no activity decrease for trypsin
and chymotrypsin. Hee et al. (1988a) stored their pancreatic juice samples at 5 °C and
analyzed them within 12hours of collection. During this period enzyme activities (amylase,
lipase, trypsin, chymotrypsin) did not decrease.
Low (1982) analyzed digesta samples for trypsin, chymotrypsin and peptidases within one
month of collection. He found that storage at -20 °C for less than three months did not
influence enzyme activities in the samples.
Howard and Yudkin (1963) analyzed pancreatic tissue homogenates after storage for one
night in a refrigerator. Enzyme activities (trypsin, amylase, proteinase) had not diminished.
Magee and Hong (1959) found that amylase and protease activities in pancreatic juice
decreased when the pancreatic juice was kept at room temperature. Amylase and trypsin
activities decreased quickly in contaminated pancreatic juice.
Legg and Spencer (1975) studied the stability of pancreatic enzymes in human duodenal
fluid to storage temperature and pH. They found that the activity of amylase, trypsin and
lipase remained relatively constant over a 4week period when stored at -20°C.Storage at
room temperature caused a considerable decline inenzyme activities,while storage at 4°C
gave intermediate results. At pH < 5, enzyme activities diminished substantially within 4
weeks of storage at -20°C.
Corring (personal communication, 1988) recommended storage of pancreatic juice at -80
°C to prevent loss of enzyme activities.
Gorrill and Thomas (1967) and Reboud et al. (1962) described enzyme analysis quite
extensively, with special reference to activation influences (see Figures 3 and 4).
It is clear that biochemical reactions in stored biological samples can influence enzyme
activities depending on storage conditions.
The effects of different storage conditions on enzyme activities in pancreatic juice should
be investigated in more detail. Storage conditions as well as assay methods should be
51
Chapter III
standardized to facilitate comparison of research data.
Regulation of PancreaticSecretion,a Feedback Mechanism
Asearlyasthe 1920s,Elmanand McCaughan(1937)establishedthefatal effect oftotalloss
of pancreatic secretions in dogs: after 2 or 3 days of total pancreatic juice deprivation by
means of a pancreatic duct catheter, the animal lost appetite, followed by vomiting and
death on day 5 to 8 after surgery.
Corring (1973, 1974) fitted pigs weighing ca 45 kg with pancreatic fistulas and duodenal
cannulas.Hefound that pancreaticsecretion isregulatedbyashort-term negativefeed back
mechanism:thepancreaticsecretion increased within2hoursofpancreaticjuice withdrawal
from the animal (volume fivefold, total protein secretion 1.5- to twofold and enzyme
activities twofolds compared to the values before juice withdrawal). Infusion of a salts
mixture (NaHCO s and NaCl, in amounts equal to pancreatic juice composition) or of
deproteinized pancreatic juice in the duodenum did not lower pancreatic juice secretion.
This indicates that the active substance in juice secretion regulation can be found in the
protein fraction of the juice. Infusion of an activated trypsin solution (even with pH < 7)
or of the collected pancreaticjuice did lower juice secretion.
From thesefindings Corring(1974)concluded thatthe(proteolytic) enzymesare responsible
for the feed back, possibly through an inhibitory action on the synthesis and release of
secretin and cholecystokinin-pancreozymin (CCK-PZ).
After perfusion (intravenously, i.v.) with secretin during half an hour the amount of
pancreaticjuice increased, while the amount of secreted protein remained constant. From
this it can be concluded that secretin can regulate the secreted amount of pancreaticjuice
without affecting the amount of protein secreted.
52
Chapter III
2
80
120
2O0
activation time frnin)
4.00
0.70
80
160
240
320
400
activation time (min)
Figure 3a.
Effect of pancreatic juice protein concentration and CaCl2 on activation of
trypsinogen by enteropeptidase at 37°C:
no CaCl2;
0.05 M CaCl2 final concentration;
+0.13 mg protein/ml; O0.27 mg protein/ml; A 0.54 mg protein/ml.
One trypsin unit on ordinate equals hydrolysis of 1 /jmole TAME per minute and per
mg pancreatic juice protein (Gorrill and Thomas, 1967).
Figure 3b.
Effect of pancreatic juice protein concentration and CaCl2 on activation of
chymotrypsinogen by enteropeptidase at 37°C:
no CaCl2;
0.05 M CaCl2 final concentration;
+0.13 mg protein/ml; O0.27 mg protein/ml; A 0.54 mg protein/ml.
One chymotrypsin unit on ordinate equals hydrolysis of 1/imole BTEE in reaction
mixture of 26% methanol (v/v) per minute and per mg pancreatic juice protein
(Gorrill and Thomas, 1967).
53
Chapter III
50
75
125
incubation time (min)
10
15
20
30
incubation time (hours)
Figure 4a.
Activation of chymotrypsinogen in rat pancreatic tissue. Activity measured with
ATEEasasubstrate,o incubation with trypsin at0°C;• incubation with trypsinat
35°C;(Reboud etal.,1962).
Figure 4b.
Activationoftrypsinogeninporcinepancreaticjuice.Incubationwithtrypsinat0°C;
(Reboud etal.,1962).
After glucagon injection (1 mg, i.v.) the amount of protein and the volume of juice
decreased, evenwhen the collectedjuicewasnot reintroduced tothe duodenum.Thiscould
be due to inhibition of secretin by glucagon (competition between secretin and glucagon
acting on the same receptor).
54
Chapter III
Nervous (nervus vagus) and hormonal (secretin and cholecystokinin-pancreozymin)
mechanisms control pancreatic secretion. The release of these hormones is controlled by
the secretion of the pancreas itself (mainly trypsin).
Effects ofAge on Pancreatic Secretion
Corring et al. (1978) studied the development of the pancreas and pancreatic enzymes in
young piglets.Theyfound that the development of the pancreas until 4weeks of age isdue
to hyperplasia and afterwards (from 4 to 8 weeks of age) to both hyperplasia and
hypertrophy.
In the rat and the chicken onset of amylase synthesis and development could be stimulated
by corticosteroids (Koldovsky and Kahn, 1964 and Yalovsky et al, 1969, both cited by
Aumaitre, 1972).
This was confirmed in the piglet by Baintner and Nemeth (1982) who treated suckling
piglets with triiodothyronine and prednisolone between two and three weeks of age. At
three weeks of age, the animals were killed and sampled. Amylase activity in the pancreas
and trypsin activityinthe intestinal contentswere higher intreated pigletsthan in untreated
controls. (Thymosin activity in gastric contents was lower in treated animals.
Chappie et al. (1990a,b,c) studied the influence of corticosteroids on carbohydrase
development in suckling piglets. They found that the amylase activity in pancreatic tissue
canbe substantially stimulated byglucocorticoid (hydrocortisone) injections (25mg/kgbody
weight), administered every other day for the last two weeks of suckling (Chappie et al.
1990a). ACTH injections had no effect on pancreatic tissue enzyme activity. In a further
study, Chappie et al. (1990b) found no effect of hydrocortisone administration on small
intestinal disaccharidase production in 14to 26dayold piglets.In6dayold piglets, Chappie
et al. (1990c) also found an positive effect of hydrocortisone injection on amylase activity
in pancreatic tissue. Lactase activity was enhanced by hydrocortisone treatment and by
ACTH injection. Chappie et al. (1990a,b,c) concluded that glucocorticoid levels can
stimulate digestive enzyme development in suckling piglets.
Secretion Rate
Kvastnitskii (1951) performed extensive studies on physiology of digestion in pigs. One of
the factors he investigated was the pancreatic secretion infistulated piglets from 20daysof
55
Chapter III
age.
He found in piglets from 20 to 30 days of age a secretion of 150 to 350 ml per 24 hours.
This secretion rate is similar to the values found by Pekas etal. (1960) in piglets between
4 and 5weeks of age (secretion of .0 to 11.0 ml per hour, pH between 7.8 and 8.2).
In pigs aged 200to 250 days,Kvastnitskii (1951) found a pancreatic secretion rate of 8000
to 9000 ml per 24 hours.
Harada etal.(1988) measured pancreatic secretion in suckling piglets under anaesthetized
conditions in response tovarious stimulants.Their results indicate that piglets from 3days
of age are capable of pancreaticjuice secretion stimulated byexogenous secretin and vagal
stimulation and by means of endogenous secretin through duodenal acidification.
Pekas et al. (1966) used fistulated piglets to measure pancreatic juice secretion. The
spontaneous secretion in piglets from 4weeks of age onwards was very variable. This was
probably due to Pavlov reflexes.
Pierzynowski et al. (1988) fistulated piglets at 3 days to 9 weeks of age. They found no
influence of suckling on pancreatic juice output in unweaned piglets. After weaning, the
feeding of solid food increased postprandial secretion (see Figure 5).
Westrom etal. (1989) assessed the levelsof pancreaticjuice secretion inpigletsbefore and
after weaning.They found that duringthe first four weeks oflife (before weaning) pancreas
secretion remained low (ca .5mlper hour per kgbodyweight).After weaning (at 5weeks
of age) the secretion increased to 2 ml per hour per kg liveweight by 12weeks of age.
Figure 6 summarizes data on pancreatic juice secretion in response to age in pigs.
Enzymatic Activity
Sucklingpiglets are fully adapted tothe ingestion and digestion of sow'smilk.The secretion
of lactase and lipase is well developed at an early age. Pancreatic proteolytic enzyme
secretion is not fully developed in suckling piglets. The digestion of milk proteins takes
place primarily in the stomach through the clotting of milk and the action of chymosin and
lactate. Immunoglobulins should not be hydrolyzed, they are to be absorbed from the gut
lumen in a complete form. Therefore, colostrum contains a trypsin inhibitor to prevent the
breakdown of these proteins.
Kvastnitskii (1951) assessed the enzymatic activityof pancreaticjuice.He found a decrease
in proteolytic and lipolytic activity of pancreatic juice between 20 and 200 days of age.
During this period, amylase activity remained relatively constant. The digestive capacity of
the stomach increased considerablywith age,sothat Kvastnitskii (1951) concluded that the
56
Chapter III
fully developed digestive capacity of the young piglet's pancreas compensated for the
insufficiently developed capacity of the stomach. Unfortunately, Kvastnitskii does not give
detailed information on sampling techniques or enzyme analyses.
In slaughter experiments with suckling piglets which had access to creep feed, Corring et
al.(1978)found that between 4and 8weeks ofagethetotal enzymaticactivityper pancreas
increased 4- to 20-fold.
Protein concentration in pancreatic juice increased remarkably in response to CCK-8
injection after one week of age in anaesthetized piglets. The ratio of amylase to protein
(mU//ig) was 80 mU/jug at 3 days old; it increased gradually from 6 to 21days of age and
afterwards itincreased abruptly to 300mU/jtg at 28daysof age (Harada etal, 1988).These
findings agreewith theresults of Hudman etal.(1957),whofound verylowamylase activity
per gram of drypancreas in one dayold piglets.The amylase activityincreased sharply until
day 28.After day 28amylase activity stayed relatively constant. Pancreatic amylase activity
per piglet (per total pancreas) increased until day 35. Between day 14 and day 21 this
increase was relatively small; during this period the pancreatic weight increased sharply
while the amylase activity per gram of pancreas remained relatively constant.
According to Pekas et al. (1966), the activities of protease, amylase and lipase in the
pancreaticjuice of pigletsaged 4to 7weeksvariedwidelyandwith no apparent association
between them.
Gorrill and Friend (1970) studied enzyme activities in pancreases from piglets between 3
and 5weeks of age. They did not find a distinct relationship between proteolytic enzyme
activity in pancreatic tissue and in the ileal digesta. Trypsin activity increased much more
than chymotrypsin activity between 3 and 5weeks of age.
Enzyme activity and especially changes herein are determined by developmental stage at
birth and byfeed composition (Baileyetal, 1956).Therefore it canbe expected that there
are considerable variation between animals with respect to enzymatic development.
Westrometal.(1987)studied the development ofpancreatic enzymesinporcine pancreatic
tissue from the fetal period to adulthood. They found an quantitative change in enzyme
activities as well as a qualitative change: enzyme activities in activated pancreatic tissue
increased duringthefetal period, after birth a decrease inenzymeactivities occurred during
ca one week and afterwards enzyme activities increased further, especially between 10 to
14weeks and 6months of age. Itwas also shown that after birth 'new' types of proteinases
developed and 'fetal' types disappeared during the first 14weeks of life.
Creep feeding of piglets before weaning can play an important role in the development of
57
Chapter III
pancreatic enzymes with age:
Creep feeding of piglets caused increased pancreatic amylase activity. At 10weeks of age
there was no measurable effect of creep feeding on pancreatic amylase activity (Shieldset
al, 1980).
After weaningat 4weeks of age (Lindemann etal, 1986)a depression occurred in amylase,
lipase, trypsin and chymotrypsin activity during the first week after weaning. There was
however no effect on gastric proteases.
Hartman etal.(1961) found no influence ofweaning (at 1 week of age) on amylase activity
in piglets' pancreatic tissue between 1 and 8 weeks of age. After weaning, lipase and
proteinase activities in pancreatic tissue declined during the first week after weaning.
Hartman etal.(1961)found no significant difference inenzyme activities of gastrointestinal
chyme between adult pigs and 1to 2 month old piglets.
In a pig of 50kg ca 10gof enzymatic (endogenous) protein is secreted with the pancreatic
juice every day (Corring and Jung, 1972).
X
a
£
1
Figure 5.
Typicalpancreaticjuicesecretion obtainedbefore (unfilled symbols)anafter (filled
symbols)feeding inexperimentsonthesamepigbefore(a,*)andafter (<>,•)weaning.
(Pierzynowski etal.,1988).
58
Chapter III
suckling
Figure 6a.
weaned
growing
Pancreatic secretion in pigs of different ages.
400
4 A
*
O OA
+
t
A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
references
Figure 6b.
Pancreatic secretion (volume, ml/h).
30
A B C D E F G H I
J K L M N O P Q R S T U V W X Y Z
references
Figure 6c.
Pancreatic secretion (protein, mg/ml).
59
Chapter III
A
B
C
D
E
F
G
H
I
J
K
L
M
O
Q
R
S
T
U
V
W
Y
+
A
high amount
of TI
0%CP
milk protein
10kg LW
16% CP
juice
reintroduced
low fiber
low amount
of TI
30% CP
soy protein
10kg LW
NaClinfusion
15%CP
2% CFat
idem
noWB
0%CP
untoasted
soy
St/Su/C
B/W/FM
review
St/C
SBM
control
suckling,
2.5 kgLW
St
one meal
weaned,
15kgLW
Legend to Figure 6b and 6c.
O
+
juice
reintroduced
high fiber
alphafloc
glucoseinfusion
15%CP
10% CFat
idem
40% WB
16%CP
SBM
toasted
soy
B/SBM
W/WB/C
B/SBM
canola meal
high fat
suckling,
8kgLW
10%
cellulose
two meals
10%CP
40% CP
juice
reintroduced
high fiber
straw
juice
reintroduced
•
juice not
juice not
reintroduced reintroduced
0%CP
2% CFat
idem
16%CP
'supro 620'
W/WF/C
WF/C
high starch
weaned.
8kgLW
10%
strawmeal
three meals
weaned,
13kgLW
7.5%
pectin
TI = trypsininhibitor, CP = crudeprotein, CFat = crudefat, C~ casein,FM= fishmeal,SBM — soybeanmeal,Su= sucrose,
St = starch, B = barley, W = wheat, WF = wheat flour, WB = wheat bran.
A=Oiimek and Sauer (1985), B=Corring and Saucier (1972), C=Pekas et al. (1966), D=Corring et al. (1972), E=Corring
(1974), F=Maenhout (1988), G-Simoes Nunes (1982), H=Hee et al. (1988a), I=Hee et al. (1985), J=Langlois et al. (1987),
K=Oiimek et al. (1984), L=Schumann et al. (1983), M=Zebrowska et al. (1983), 0=Partridge et al. (1982), P=Zebrowska
(1985), Q=Corring (1979), R=Zebrow»ka et al. (1984), S=Imbeah et al. (1988), T=Corring and Chayvialle (1987),
U=Pienynowski et al. (1988), V=Mosenthin et al. (1988), W=Hee et al. (1988b), X=Wass (1965), Y=Ihse and Lilja (1979),
Z=Harada et al. (1988)
60
Chapter III
Conclusions
From the data summarized herein itisclear that pancreatic secretion (volume,protein and
enzyme activities) develops with increasing age of the animal. Alterations in feeding
regimen, feed intake and diet composition (e.g., at weaning) interacts with this agedependency.
Pancreatic fistulation is difficult to perform in small animals, so that not many data are
available on pancreaticjuice secretion inyoung piglets.Furthermore, enzyme development
also depends on creep feed intake,weaning age and composition ofweaner diet. Therefore
itisdifficult toobtain aclear picture of pancreatic secretion inyoungpiglets.More research
in this field is needed.
Data on pancreatic secretion in older pigs (sows and boars) are not known.
After Kvastnitskiis experiments described in his thesis (1951),no further long-term studies
have been made on the changes in pancreatic juice secretion with age.
Effects of FeedIntake and Feeding Frequency on Pancreatic Secretion
Secretion Rate
Corring et al. (1972) studied the effect of a meal on pancreatic secretion. They used
fistulated animals of ca 40 kg.
They found a strongincrease in pancreatic protein excretion after a meal;protein secretion
stayed high for 5 to 7 hours, showing a peak about 3 hours after starting the meal. This
phenomenon canbe explained bythenegative feed backmechanism discussed inaprevious
paragraph: during the postprandial period, secreted enzymes form complexes with the
ingested substrate sothat theyare nolonger availablefor the down-regulation of pancreatic
secretion. As soon as the substrate ishydrolyzed the enzyme isliberated from the enzymesubstrate/product-complex and down-regulation of secretion is resumed.
In suckling piglets, this phenomenon is not found (Pierzynowski et al, 1988) since feed
intake during suckling occurs almost continuously (ca 24 meals a day).
Pancreaticjuice secretion depends onthe frequency offeeding: Heeetal.(1988b)used four
barrows to investigate this phenomenon. They found an increase in daily secreted juice
volume of .5 liter for each additional meal (feeding 1to 3 times daily).
61
Chapter III
Enzymatic Activity
Corring and Jung (1972) noted that with a fixed diet the amino acid composition of the
pancreatic juice of pigs is constant.
From this it can be concluded that the enzymes ratio in pancreatic juice does not change
during the daywhen a fixed diet isfed to adapted pigs.Changes in enzyme activities during
the day occur only in terms of total amounts of enzymes, the relative proportion of e.g.,
trypsin remains the same.
The amount of secreted protein per 24 hours is not related to the feeding frequency
(number of meals per day):when feed intake was 900 gram per day (1x 900 or 2x 450,
protein in feed = 16.86 %) 5 to 7 grams of protein was secreted in the pancreatic juice.
Hee et al. (1988b) found with increasing feeding frequency (one, two or three meals daily)
that the amylase secretion increased two-fold with each additional meal. The lipase
secretion was highest when the pigs were fed once daily. The secretion of protein, trypsin
and chymotrypsin was not affected.
Adaptation of Enzyme Activities to Diet Composition
Digestive enzymes adapt to the amount of substrate in the feed (Corring, 1980).
When changing from milk to starch rich feed (at 35 days of age) a sharp increase in
amylase activity occurs in piglets 7 days after the diet change (Aumaitre and Rerat, 1966,
unpublished data, cited by Aumaitre, 1972).
The adaptation to a new diet takes 5 to 7 days in rats and growing pigs.After this period
a new, stable enzyme status has been established (Ben Abdeljlil and Desnuelle, 1964;
Corring, 1980).
Chymotrypsin activitystarts changing 48hours after a diet change and reaches anew stable
level at 5 days after the diet change (Corring and Saucier, 1972).
The adaptation to diet composition (5 to 7 days) takes much longer than the feedback
regulation (2 to 3 hours) discussed earlier. The feedback mechanism responds directly to
the amount of enzymes present in free form in the gutlumen. This 'short term' feed back
mechanism ensures an appropriate flow of enzymes into the gut lumen when feed isgiven
to adapted animals.
The relatively slow reaction of pancreatic secretion in response to feed composition could
be explained by other physiological changes occurring after a diet change, such as gastric
62
ChapterIII
emptying rate and gut motility.
The mechanism of nutritional regulation of pancreatic function (biosynthesis as well as
secretion of enzymes) isnot completely clearyet.In 1977,Corring reviewed the adaptation
of pancreatic enzymes to dietary components. The hydrolysis products of dietary
components stimulate enzymatic secretion through release of CCK-PZ, secretin, insulin
and/or other gastro-intestinal hormones or peptides.
To elucidate the nutritional regulation of pancreatic secretion, Corring and Chayvialle
(1987) measured hormones and peptides in the blood of pigsfed on different diets (control
vshigh-starch or high-fat) to check the influence of these substances on pancreatic enzyme
secretion. Pancreatic enzyme secretion (amylase and lipase) clearly adapted to the amount
of substrate inthe feed (starch and fat respectively).With allthree diets a postprandial rise
inplasma cholecystokinin, somatostatin and pancreatic polypeptide wasfound inthe blood.
Plasma secretin levels did not alter after feed intake.
Corringand Chayvialle(1987) found that plasma levelsofsecretin and cholecystokinin were
not affected by an increase in fat or starch intake. Thus,these two hormones do not seem
to play an important role in the nutritional regulation of the lipase or amylase secretion by
the pancreas.
Corring (1980) discussed the physiological significance of the adaptation of digestive
enzymes to the diet: Enzyme secretion is sufficient to digest much larger amounts of
substrate than are normally ingested, when enzyme activities measured in vitro are
compared with feed components intake in vivo. However, the digestion of a substance in
vivo could necessitate a higher enzyme concentration over a certain period, because rate
of hydrolysis depends on enzyme concentration. Rate of hydrolysis is important in the
digestive tract of animals where substrates are transported along the gut.
The ability of enzyme secretion to adapt to diet composition eliminates the need for a
continuously high secretion rate, which would cause unnecessary losses of (unabsorbed)
endogenous protein.
Corring (1980) also refers to experiments with rats performed by Le-Thanh Uyen (1969,
cited by Corring, 1980) inwhich prolonged protein malnutrition caused an increase in the
secretion of proteins (enzymes) with pancreatic juice. This hypersecretion is possibly a
source of protein substrate for the animal.
The physiological backgrounds of pancreatic adaptation mechanisms are not fully
understood yet.
63
Chapter III
Effects of Diet Composition on Pancreatic Secretion
Secretion Rate
Pekas et al.(1966) found a twofold pancreaticjuice secretion in piglets (age at surgery: 45
days)fed on (heat treated) soya protein compared topigletsfed onmilkprotein.Thiscould
be caused by (heat stable) trypsin inhibitors in the soya feed.
Zebrowska etal. (1983) performed experiments with growing pigs (ca 35kg) on two diets:
diet A (purified): casein, wheat starch, sucrose, soya oil and diet B (cereals): barley meal,
soybean meal. With diet B they found higher pancreatic juice secretion (2182versus 1204
g per 24 hours), but no difference in enzyme activities.
The same effect of feed composition was found by Partridge et al. (1982). They fed two
diets to growing pigs:diet A (purified): starch, sucrose, casein, maize oil,cellulose and diet
B (cereals): barley, wheatings, fish meal. Diet A caused a pancreatic flow of 1273 ml per
24 hours, diet B a flow of 4962 ml per 24 hours.
Corring (1980) found with cereal diets in pigs of ca 45kg a pancreatic flow of 2500 ml per
24 hours.
Table 1.
Effect of diet composition onpancreatic juice flow (Zebrowska, 1985)
diet
I
wheat
wheat bran
wheat flour
casein
wheat starch
vit/min-mix
cellulose
flow rate
pancreatic juice
88.7
7.0
4.3
4108
II
III
IV
44.4
44.3
3.5
3.5
4.3
-
44.4
44.3
6.0
1.0
4.3
-
.
85.7
6.0
4.3
4.0
4560
2556
64
1757
%
%
%
%
%
%
%
ml/24h
Chapter III
Zebrowska (1985) found with pigs of ca 34 kg an effect of diet on pancreatic flow (see
Table 1). Secretion of pancreatic juice was higher in pigs fed diets containing wheat or
wheat + wheat bran than in pigs fed wheat flour or wheat flour + wheat.
In Table 2 some data on pancreatic juice secretion in growing pigs in response to diet
composition are summarized.
Enzyme Activity
The pancreatic enzyme levels depend on feed composition (protein, fat, carbohydrates,
fiber) (Grossman et al, 1943;Lindemann et al, 1986).
Wheat bran caused inrats an elevation of the amylase and trypsin activities inthe pancreas
(Schneeman et al, 1982).
In experiments with rats (Howard and Yudkin, 1963)itwasshown that amylase and trypsin
activities in pancreatic tissue were influenced independently from one another by changes
in the quantity or quality of carbohydrates or proteins in the feed.
Proteinin thefeed. Snook and Meyer (1964) measured the enzymatic activityof the
digesta in relation to the protein content of the diet. They suggested that the protease
activityis enhanced through an increase in the synthesis and secretion of digestive enzymes
and a decrease in the rate of enzyme breakdown in the digestive tract.
AccordingtoPekasetal.(1964)thesoyprotein digestion depends on (pancreatic) enzymes;
this dependency decreases with increasing age of the animals.
In ca 7weeks old piglets, protease, amylase and lipase secretion was about 5 times higher
when (heat treated) soya protein was fed compared to milk protein (Pekas et al., 1966).
The biosynthesis of proteolytic enzymes in the pancreas increases when untreated soya
(containing trypsin inhibitor) is fed (Corring, 1980).
Schumann et al. (1983) investigated the pancreatic secretion in growing pigs (ca 35 kg) in
relation to the quality of the protein source in the diet.Theyfound ahigher pancreaticjuice
secretion in pigs fed a diet with untoasted soybean meal (solvent extracted) compared to
pigsfed a diet with toasted soybean meal.The protein secretion over 24hourswas elevated
with the untoasted soya-diet, as was the trypsin, chymotrypsin, amylase and to a lesser
extent the lipase activity.Schumann etal.(1983) explained these effects bythe presence of
a trypsin inhibitor in untoasted soya, which blocks the trypsin-mediated feed back to the
pancreas.
65
Chapter III
Table 2.
Pancreatic secretion inpigs adapted todifferent diets per24hours
Reference
Feed intake
(kg diet)
Vol.
(1)
Prot.
(g)
Corring et al.
1972
Corring1980
Zebrowska et al.
1981
Partridge et al.
1982
Schumann et al.
1983
Zebrowska etal.
1983
0.9 barley-soya
1.7
1.0 barley-soya
1.5 starch-casein
1.5 barley-soya
1.6barley-fish meal
1.6 starch-casein
0.9 rawsoybean meal
0.9 toasted soybean meal
1.5 barley-soya
1.5starch-casein
1.8 starch-soya
1.7 protein-free
1.7soybean meal
1.7isolated soyprotein
1.7raw soy-product
1.7autoclavedsoy
1.8high fat
1.8 control
1.8 protein-free
1.4 wheat
1.4wheat/wheat bran
1.4wheat/wheat flour
1.4 wheat flour
1.6nowheat bran
1.640%wheat bran
1.8control
1.8 high fat
1.8 protein-free
1.6soybean meal
1.6canola meal
1.8 starch
1.8 cellulose
1.8straw meal
1.8 pectin
2.5
1.2
2.2
5.0
1.3
2.4
1.5
2.2
1.2
3.8
3.9
4.0
3.9
4.0
3.6
3.9
4.0
3.5
4.1
4.6
2.6
1.8
1.7
3.6
4.0
3.9
3.5
1.2
1.0
3.7
3.2
4.6
4.7
Ozimek etal.
1984
Ozimek etal.
1985
Hee et al.
1985
Zebrowska etal.
1985
Langlois etal.
1987
Hee et al.
1988a
Imbeah etal.
1988
Mosenthin et al.
1988
Live
weight
Typeof
cannula
6.1
42 kg
duct
18.6
10.9
12.1
9.8
6.7
9.4
4.7
12.1
10.9
14.4
19.0
28.6
28.9
23.6
19.4
15.3
15.0
12.2
17.9
19.0
15.8
13.0
14.6
19.7
15.1
15.3
13.1
16.4
15.4
26.9
22.8
28.5
27.0
45
35
35
48
48
35
35
40
40
35-50
35
35
35
40
40
35-100
35-100
35-100
34
34
34
34
38
38
35
35
35
47
47
60
59
69
72
duct
pouch
pouch
duct
duct
duct
duct
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
duct
duct
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
pouch
According to Pond et al. (1971) the difference in performance in favor of piglets fed diets
with ISP (Isolated Soybean Protein) compared to animals fed on FPC (Fish Protein
Concentrate) can not be explained by differences in pancreatic enzyme activities. Enzymes
66
Chapter III
of the stomach or gut wall could also play a role in this respect.
In conclusion, protein degrading enzymes respond to the amount of protein as well as the
type of protein in the feed. Trypsin inhibitors in the feed can induce hypersecretion of the
pancreas by blocking the feedback mechanism controlling secretion.
Fatinthefeed.The specific lipase activityinpancreaticjuice ofpigsincreasesby700
% when the triglyceride intake is increased from 30 to 220 g (Corring, 1980). It is not
unlikely that lipase synthesisismore stimulated byunsaturated fatty acidsthan bysaturated
fatty acids.Furthermore theratiobetween carbohydrates,proteins and fats inthe diet could
influence lipase activity, as could the relative amounts of carbohydrates and fats in terms
of energy (Corring, 1980).
Ozimek et al. (1985) also investigated the effect of fat quantity and quality on lipase
secretion in the pig. When raising the amount of fresh canola oil in the diet from 0 to 15
%, lipase secretion increased from 2.96 x 10s U per 24 hours (when no canola oil was
added) to 10.17x 10sU per 24hours.The inclusion of 15% peroxidized canola oil resulted
in an 6 to 9 fold increase in lipase activity.
Forman and Schneeman (1980) studied the effect of pectin and fat on the exocrine
pancreas in rats. They found an increase in the activity of lipase and a decrease in the
activities of amylase and to a lesser extent of trypsin and chymotrypsin when the amount
of maize oil in the diet was increased from 5to 25 %. An effect of pectin was found only
on enzyme activities in the small intestine.
The amount of fat in the feed as well as the quality (degree of saturation, chain length)
affects lipase secretion in pigs.
Starch in thefeed. Increasing the amount of starch in the diet from 160 to 600 g
resulted in an increase of the specific amylase activity (Corring, 1980).
Effects of different types of starch (maize, potato, wheat, raw vs cooked) are not known.
Conclusions. From these data it is clear that pancreatic enzyme secretion adapts
specifically to the substrates available from the ingested food.
67
ChapterIII
Effects of Environment and Stresson Pancreatic Secretion
Enzymatic Activity
Szabo et al (1976) found that total amylase and lipase content in pancreatic homogenate
were influenced by environmental temperature: concentrations were low at high
temperatures (38 - 40°C,relative humidity 50 %) and high at low temperatures ( 0 - 2 °C,
relative humidity 90 %) compared to control temperature (25-27°C,relative humidity 60
%). Inthese experiments feeding wasad lib,sothat feed intake could have affected results.
Discussion
Snook (1974) assumes that in healthy, normally fed animals it is unlikely that changes in
enzymelevels(induced bydietcomposition) haveasignificant influence onthe physiological
well-being of the animal.
Several workers stress the discrepancy between the excessive production of enzymes and
the adaptation of enzyme activities to different diets (Low, 1982; Partridge et al, 1982;
Zebrowska et al, 1983). The amount of digestive enzymes secreted by the pancreas is
theoretically sufficient to hydrolyze 100times the ingested amount of feed (substrate).
Not much is known about the dilution and breakdown of enzymes in the digestive tract.
According to Snook (1965) probably more than 90 % of trypsin and chymotrypsin is
inactivated byproteolytic enzymes ofthegutwall andbymicro organisms.Itispossible that
starch can reduce the hydrolysis of amylase because starch + amylase as a complex cannot
be broken down by proteolytic enzymes.
Much research has been done on influences of diet composition on pancreatic enzyme
secretion. These data should be combined with digestibility data to gain knowledge on the
physiological significance of adaptation mechanisms. Other factors influencing pancreatic
secretion should also be taken into account.
More work should be done on enzyme development inyoung pigletsin relation to weaning
and diet composition.
Another point of interest isthe breakdown of secreted enzymes in the digestive tract ("half
life time") (De Lange, pers. commun. 1988). This was investigated by Asche et al. (1987).
They studied the molecular weight profiles of the soluble proteins in the digestive tract of
68
Chapter III
pigletsand compared themwiththemolecularweightsofthedigestiveenzymes.These data
suggested that most of the endogenous proteins were hydrolyzed rapidly.
Comparisons should be made between secreted amounts of enzymes and enzyme activities
in (ileal) digesta and faeces.
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and quantitative adaptation of exocrine pancreatic secretions.Agric. ForestryBull,
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Partridge, I.G., Low, A.G., Sambrook, I.E., Corring, T., 1982.The influence of diet on the
exocrine pancreatic secretion of growing pigs.Br.J.Nutr.48:137-145
Pekas,J.C., 1965.Permanent physiologicalfistula of thepancreas andother digestiveglands.
/. Appl. Physiol. 20(5):1082-1084
Pekas, J.C., Hays, V.W., Thompson, AM., 1964. Exclusion of the exocrine pancreatic
secretion: effect on digestibility of soybean and milk protein bybaby pigs at various
ages./. Nutr. 82:277-286
72
ChapterHI
Pekas, J.C., Hays, V.W., Thompson, A.M., Jones, J.D., Speer, V.C., 1960.A method
for the studyof pancreatic secretion inthebaby pig./.Anim. Sci. 19:1333(Abstr.311)
Pekas, J.C., Thompson, A.M., Hays,V.W., 1966.Characteristics of the exocrine pancreatic
secretion of the young pig./. Anim. Sci. 25:113-121
Pierzynowski, S.G., Westrom, B.R., Karlsson, B.W., Svendsen, J., Nilsson, B., 1988.
Pancreatic cannulation of young pigs for long-term study of exocrine pancreatic
function. Can.J.Anim. Sci. 68:953-959
Pond, W.G., Snook, J.T.,McNeill,D.,Snyder, W.I.,Stillings,B.R., 1971.Pancreatic enzyme
activities of pigs up to three weeks of age./. Anim. Sci.33(6:1270-1273
Reboud, J.P., Ben Abdeljlil, A., Desnuelle, P., 1962.Variations de la teneur en enzymes du
pancreas de rat en fonction de la composition des regimes.Biochim. Biophys.Acta
58:326-337
Schneeman, B.O.,Richter, B.D.,Jacobs, L.R., 1982.Response to dietarywheat bran in the
exocrine pancreas and intestine of rats./. Nutr. 112:283-286
Schumann, B., Souffrant, W.-B., Matkowitz, R., Gebhardt, G., 1983.Untersuchungen zur
endogenen N-Sekretion im Pankreassekret beim Schwein. Wiss. Z. KarlMarxUniv.
Leipzig;Math.-Naturwiss.R 32(6):570-575
Shields,Jr R.G., Ekstr0m, K.E., Mahan, D.C., 1980.Effect ofweaning and feeding method
on digestive enzyme development in swine from birth to ten weeks./. Anim. Sci.
50(2):257-265
Snook, J.T., 1965.Dietary regulation of pancreatic enzyme synthesis, secretion and
inactivation in the rat./. Nutr. 87:297-305
Snook, J.T., 1974.Adaptive and non-adaptive changes in digestive enzyme capacity
influencing digestive function. Fed.Proc. 33:88-93
Snook, J.T., Meyer, J.H., 1964. Response of digestive enzymes to dietary protein./. Nutr.
82:409-414
Szabo, J., Szabo, P.R., Rafai, P., 1976.Activity of pancreatic hydrolases in pigs kept at
different temperatures (in Hungarian). MagyarAllatorvosok Lapja 31(5):325-328.
Englishsummary: Nutr~Abstr.Rev. 47,no 3042,1977.
Wass, W.M., 1965.The collection of porcine pancreatic juice by cannulation of the
pancreatic duct.Am. J. Vet.Res. 26(114):1106-1109
Westrom, B.R., Ohlsson, B., Karlsson, B.W., 1987. Development of porcine pancreatic
hydrolases and their isoenzymes from the fetal period to adulthood. Pancreas
2(5):589-596
73
ChapterIII
Westrom, B.R., Pierzynowski, S.G., Karlsson, B.W., Svendsen, J., 1989. Development
of the exocrine pancreatic function: response to food and hormonal stimulation in
pigs from birth up to after weaning.In: Proceedings of the 4—International Seminar
onDigestivePhysiologyinthePig(eds. Buraczewska etal.).Jablonna,Poland,pp36-43
Zebrowska, T., 1985.The influence of the level and source of fiber in the diet on the
exocrine pancreatic secretion in growing pigs.In:DigestivePhysiology in thePig. Eds
Justet al.Beretningfra Statens Husdyrbrugsforsog 580, pp 152-154.
Zebrowska, T., Low, A.G., Zebrowska, H., 1984.Digestion of protein and carbohydrate in
the stomach and secretion of pancreatic enzymes in growing pigs.In:Proceedings of
the Vlth International Symposium on Amino Acids (eds. Zebrowska et al). Serock,
Poland,1981.PolishScientificPublishers, Warszawa, Poland, pp 388-390.
Zebrowska, T.,Low,A.G., Zebrowska, H., 1983. Studies on gastric digestion of protein and
carbohydrate, gastric secretion and exocrine pancreatic secretion in the growingpig.
Br.J.Nutr. 49:401-410
74
ChapterIV
STORAGE OF PORCINE PANCREATIC JUICE:EFFECT ON ENZYME ACnVITIES
Caroline A. Makkink1, Lucette A.J. van der Westerlaken1, Leo A. den Hartog1,3
Martin J. van Baak2 and Joop Huisman2
1
2
Agricultural University, Department of Animal Nutrition,
Haagsteeg 4,6708PMWageningen, the Netherlands
TNO-Institutefor Animal Nutrition and Physiology(ILOB)
P.O.Box 15,6700AAWageningen, The Netherlands
3
Present address:Research Institute for Pig Husbandry,
P.O.Box83,5240ABRosmalen,The Netherlands
Published in: Journal of Animal Physiology and Animal Nutrition 63 (1990) 267-272
Reproduced by permission of Paul Parey Verlag, Hamburg
75
Chapter IV
Summary
A total of 80 samples of porcine pancreatic juice were stored at three different
temperatures (4 °C, -20°C and -80 °C) for different periods of time (0 to 21 days).
Activities of pancreatic enzymes (trypsin, chymotrypsin and lipase) have been measured in
the stored pancreatic juice.
The results show that enzyme activities are affected by storage temperature.
From one week after sampling, activities of trypsin, chymotrypsin and lipase in pancreatic
juice samples stored at 4°Cdiffer from activities measured immediately after sampling (at
day0) (p < .05).Infrozen pancreaticjuice samples,enzymeactivitiesremain fairly constant
during three weeks of storage.
Introduction
Pancreatic enzyme secretion in pigs has received much attention from several animal
research workers (Partridge et al, 1982;Hee et al, 1988; Pierzynowski et al, 1988).
Fistulation techniques for pancreaticjuice collection in vivohavebeen developed (Corring
etal, 1972;Hee etal, 1985;Pierzynowski etal, 1988).Thus secretion rates over extended
periods of time and in response to different diets can be assessed.
Usually, the collected juice cannot be analyzed immediately: the samples must be stored
after collection. Unfortunately, storage conditions are often not published when
experimental results are given.
Corring (pers. comm., 1988) advises storage at -80 °C to prevent enzyme activity decline.
Different workers use different methods to store their pancreatic juice samples from
collection until analysis (see Table 1).
The purpose of this experiment was to determine the effect of different storage
temperatures on enzyme activities in pancreatic juice.
76
ChapterIV
Table 1.
Storage of pancreatic juice bydifferent authors.
Reference
(Author, Year)
Storage conditions
Time
Temp.
Reboud et al., 1962
Partridge et al., 1982
Langlois et al., 1987
Hee et al., 1988
Imbeah et al., 1988
Pierzynowski et al., 1988
months
<8h
?
< 12h
?
?
-20 °C
4°C
-80 °C
5°C
-30 °C
-20 °C
Comment
rats, lyophilized juice
pigs
pigs
pigs
pigs
pigs
Materialsand Methods
Animals. Diets. Housing and Sampling
A castrated male pig (age 14 months), fitted with a pancreatic cannula according to the
method of Heeetal.(1985)wasused for collection ofpancreaticjuicefrom asmall isolated
segment of duodenum. The method of Hee etal.(1985)was slightlymodified: the one-way
valve at the re-entrant part of the cannula caused blockage of the cannula and was
therefore removed.
The pig was fed a commercial pig diet (NE 10000 kJ/kg, 18.2 % CP, 1.21 % dig. Lysine,
0.73 % dig. Methionine/Cysteine) twice daily at 0800 and 1700 hours, 1000 g each meal.
At five different days a sample of pancreatic juice was taken.
The samples were taken at 1300 hours, five hours after feeding, when pancreatic juice
production was at its maximum in this pig (Wichers, 1988).
Each sampling took five to ten minutes and ca 30mlofjuicewas collected during thistime.
An aliquot of each sample was analyzed for enzyme activities within five hours after
sampling.
The remainder of each samplewas divided in three subsamples:one subsample was stored
at 4°C,one subsample was stored at -20°C and the third subsample was stored at -80°C.
The stored subsamples were divided intofive portions (one portion for each dayof analysis
after storage) so that no repeated freezing and thawing took place. All stored subsamples
were tested for activities of trypsin, chymotrypsin and lipase after the designated storage
period (1,4, 7, 14 or 21days).
A total of 80 aliquots was analyzed for enzyme activities.
77
Chapter IV
Analytical and Statistical Procedures
Enzyme analysis on day 0 was completed within five hours from sampling. Samples were
kept on ice from sampling until analysis was completed.
Frozen samples were left to thaw at room temperature and put on ice immediately after
thawing had been completed.
Trypsin activitywas measured with Na-p-toluolsulphonyl-L-argininmethylester (TAME) as
a substrate (Bergmeyer, 1974).
Chymotrypsin activity was measured with N-benzoyl-L-tyrosinethylester (BTEE) as a
substrate (Bergmeyer, 1974).
Lipase activitywasmeasured using a method developed byVan Oort (not published, 1988)
with l-thio-2,3-tributyryl-glycerol (not commercially available) as a substrate: the substrate
ishydrolyzed to free fatty acids and a glycerol-like substance with a free SH-group. DTNB
(5,5-dithiobis(2)-nitro-benzoic-acid) isadded toform acolored product onreactionwiththe
previously formed SH-group.
Aspectrophotometer (Perkin-Elmer,type550)wasused tomeasure theextinction changes.
Extinction changes were recorded on paper to facilitate calculations.
Pancreaticjuicewasdiluted 10to 100-fold with a buffer solution (depending onthe enzyme
and on the amount of enzyme activity present in the sample).
All analyses were carried out at 25°C, trypsin analysis at pH 8.1,chymotrypsin analysis at
pH 7.8 and lipase analysis at pH 8.5.
No activation of zymogens was performed prior to analysis of trypsin and chymotrypsin.
Enzyme activities were also calculated as a percentage of the activity on day 0 (reference
value).
Data were subjected to analyses of variance according to the following model:
Yijk = ju + SUBSAMPLEi(k) + DAYj + DAY*TEMP (j , k) + eijk
In which:
SUBSAMPLE, /kj
- dependent variable
- overall mean
- subsample nested within temperature
(TEMP= 4°C:i = 1,2,3,4,5)
(TEMP=-20 °C: i = 6, 7, 8, 9, 10)
(TEMP=-80 °C: i = 11, 12, 13, 14, 15)
78
Chapter IV
DAYj
DAY*TEMPj»k
eijk
- days after sampling, storage period
G = 0, 1,4, 7, 14, 21)
- interaction between storage period and temperature
- error term
The effects of DAY, SUBSAMPLE and DAY*TEMP were tested against the error term.
All tests were performed using SAS-GLM (SAS 1985).
Post hoc analyses of the interaction between DAY and TEMP were performed using
Student's T tests (SAS-GLM: SAS, 1985).
Results
Activities of trypsin, chymotrypsin and lipase are presented in Tables 2, 3 and 4,
respectively. Relative activities (percentages of activity on day 0) of trypsin, chymotrypsin
and lipase are shown in Figures 1, 2 and 3, respectively.
No differences in enzyme activitywere found (p > .05) between storage at -20°Cand at 80 °C.When stored frozen, enzyme activities did not change over the three weeks storage
period (p > .05).
Between day 0 and day 1,trypsin activity showed a small increase which was however not
statistically significant.
After day 0, trypsin activities in frozen samples ranged from 119to 144 % of the activity
found on day 0, chymotrypsin activities in frozen samples ranged from 88 to 107 % and
lipase activities in frozen samples from 46 to 108 %.
All enzyme activities decreased in samples stored at 4°C.
The enzyme activities of samples stored at 4°Cdiffered from the initial enzyme activity (p
< .05) from 14, 4 and 7 days of storage onwards for trypsin, chymotrypsin and lipase
respectively.
After three weeks of storage at 4°C enzyme activities were reduced to 31, 39 and 5 % of
the activities on day 0 for trypsin, chymotrypsin and lipase respectively.
79
Chapter IV
Table 2.
TEMP
4°C
-20°C
-80°C
Trypsin activities in pancreatic juice stored at different temperatures from 0 to 21
days (Least Square Means x ), units per ml.
DAY
0y
14
1
47.71aA
47.7"
47.7b
62.5^
60.5^
58.0 abA
51.5^
62.2 aA
59.6 abA
35.0 abA
61.1 a B
68.6 aB
219
21
bcA
57.2 aB
64.6 abB
14.TcA
64.3 a
56.7a
Least Square Means within a row (abc) orcolumn (AB) that do not have a common superscript differ (T test, p <
.06). R 2 = 0.91, RSD = 14.48
On day 0, samples had not been stored prior to analysis, all analyses performed with samples on ice.
Chymotrypsin activities in pancreatic juice stored at different temperatures from 0
to 21 days (Least Square Means x ), units per ml.
Table 3.
TEMP
4°C
-20 °C
-80 °C
DAY
0"
31.9 aA
31.9^
3j 9 abA
1
30.0 abA
33.6^
34.2^
4
7
25.2 bcB
32.9^
32.1 abA
214
14
cdB
29.5^
30.6 abA
21
16.5 deB
309
aA
30.0 abA
12.5eB
29_4aA
28.0 bA
Least Square Means within a row (abcde) or column (AB) that do not have acommon superscript differ (T test, p
< .06). R 2 = 0.90, RSD = 4.09
On day 0, samples had not been stored prior to analysis, all analyses performed with samples on ice.
Table 4.
TEMP
4°C
-20°C
-80°C
Lipase activities in pancreatic juice stored at different temperatures from 0 to 21
days. (Least Square Means x ), units per ml.
DAY
0y
21.3^
<yi oabA
21.3 abA
21
14
16.9a
22.9a
19.0a
11.5abcA
15.1abA
15.8abA
6.0
bcA
2.0
cA
123abA
g qbA
| i gabA
10.3 bA
Least Square Means within a row (abc) orcolumn (ABC) that do not have a common superscript differ (T test, p
< .05). R 2 = 0.53, RSD = 9.05
On day 0, samples had not been stored prior to analysis, all analyses performed with samples on ice.
80
1.0cC
120abBC
•yy 1aAB
Chapter IV
Figure 1.
Trypsin activity asa percentage of activity on day 0.(Least Square Means, Standard
Error of LSM = 7.8). * =Relative activity differs from 100 %(p <.05).
e
0
10
12
14
18
IB
20
22
storage time (days)
Figure 2.
Chymotrypsin activity as a percentage of activity on day 0. (Least Square Means,
Standard Error of LSM =6.5). * = Relative activity differs from 100 %(p <.0.5).
•
6
10
12
14
16
18
20
22
storage time (days)
Figure 3.
Lipase activity as a percentage of activity on day 0. (Least Square Means, Standard
Error of LSM =20.7). * =Relative activity differs from 100 %(p <.05).
81
Chapter IV
Discussion
Lipase activity showed considerable variation between measurements. Therefore, it is
advisable to analyze more samples when lipase activity is to be measured.
We did not activate the proteolytic enzymes prior to analysis.
The fact that considerably high levels of these enzymes were found in unactivated
pancreatic juice samples indicated that activation of zymogens was not necessary. Hee et
al. (1988) and Sauer (pers. comm. 1988) had to use an activation procedure to be able to
measure proteolytic enzyme activities.The fact that we did not use a one-way valve in our
cannula to prevent backflow of chyme into the pouch may have caused activation of
tripsinogen to trypsin by enterokinase. Enterokinase from the duodenal wall could have
entered the pouch or enterokinase could be secreted by the pouch wall.This enterokinase
probably activated trypsinogen after which trypsin activated trypsinogen and
chymotrypsinogen (chain reaction).
The decline of enzyme activities in pancreaticjuice stored at 4°Cis probably due to autodegradation of enzymes or to degradation of enzymes by other enzymes or by
microorganisms (contamination of juice). In frozen pancreatic juice, enzymes and
microorganisms are less active.
From the data presented here it is clear that enzyme activities in pancreatic juice are
affected by storage temperature.
From the present experiment it canbe concluded that pancreaticjuice canbe stored frozen
for at least three weekswithout deleterious effects on the activities of trypsin, chymotrypsin
and lipase.
These results are in agreement with the data reported by Legg and Spencer (1975) who
investigated the stability of pancreatic enzymes in human duodenal fluid when stored at
different temperatures.
When stored at 4°C, pancreatic juice can be stored for 7, 1and 4 days before assessment
of trypsin, chymotrypsin and lipase activity, respectively.
The small increase in trypsin activity between day 0 and day 1of pancreaticjuice samples
might be caused by activation of remaining trypsinogen bytrypsin present in the pancreatic
juice samples.However, if this is the case, then it isnot clear why this trypsinogen was not
activated in the first place.
More research is needed on changes in enzyme activity during storage of pancreatic juice
and other samples (e.g. pancreatic tissue, chyme).
82
Chapter IV
Changes in enzyme activity of pancreatic juice within one day from sampling ('short-term
changes') should also be investigated.
It is important to find optimal storage conditions for biological samples in which enzyme
activities are to be determined.
Reboud etal.(1962) stored their lyophilized pancreaticjuice samples for months at -20°C.
Lyophilization (freeze-drying) of pancreatic juice might be a method to prevent enzyme
activity changes during long-term storage.This possibility should be studied inmore detail.
When analyzing pancreatic enzyme activities, activation of proteolytic enzymes should be
performed to check if maximal activity is obtained before analysis.
Optimalization of storage conditions for pancreatic juice would improve reliability and
comparability of literature data.
References
Bergmeyer, H.U., 1974.Methoden der Enzymatischen Analyse.
ThirdEdition. Verlag Chemie, Weinheim, WestGermany.
Corring, T., Aumaitre, A., Rerat, A.A., 1972. Fistulation permanente du pancreas
exocrine chez le pore. Application: reponse de la secretion pancreatique au repas.
Ann. Biol. anim. Bioch. Biophys. 12:109:124
Hee, J.H., Sauer, W.C., Berzins, R., Ozimek, L., 1985.Permanent re-entrant diversion of
porcine pancreatic juice. Can.J.Anim. Sci. 65:451457
Hee, J.H., Sauer, W.C., Mosenthin, R., 1988.The measurement of pancreatic secretions
in the pig with the pouch technique./. Anim. Physiol, a.Anim. Nutr. 60:241-248
Imbeah, M., Sauer, W.C., Mosenthin, R., 1988.The prediction of the digestible amino
acid supply in barley-soybean meal or canola meal diets and pancreatic enzyme
secretions in pigs./. Anim. Sci. 66:1409-1417.
Langlois, A., Corring, T., Fevrier, C, 1987. Effects of wheat bran on exocrine pancreas
secretion in the pig.Reprod. Nutr. Develop. 27(5):929-939
Legg, E.F., Spencer, A.M., 1975.Studies on the stability of pancreatic enzymes in
duodenal fluid to storage temperature and pH. Clin. Chim.Acta 65:175179
Partridge, I.G., Low, A.G., Sambrook, I.E., Corring, T., 1982.The influence of diet on the
exocrine pancreatic secretion of growing pigs.Br.J. Nutr. 48:137-145
83
Chapter IV
Pierzynowski, S.G., Westrom, B.R., Karlsson, B.W., Svendsen, J., Nilsson, B., 1988.
Pancreatic cannulation of young pigs for long-term study of exocrine pancreatic
function. Can.J.Anim. Sci. 68:953-959
Reboud, J.P., Ben Abdeljlil, A., Desnuelle, P., 1962.Variations de la teneur en enzymes
du pancreas de rat en fonction de la composition des regimes. Biochim. Biophys.
Acta 58:326-337
SAS Institute Inc., 1985.SAS User's Guide: Statistics. Version 5 Edition. Cary,NCSAS Institute Inc., 1290pp.
Wichers, E.G., 1988.Pancreassecretie bij het Varken. Internalreport TNO-IGMB
ILOB-department, Wageningen,TheNetherlands,43pp.
84
ChapterV
GASTRIC PROTEIN BREAKDOWN AND PANCREATIC ENZYMEACTIVITIES
IN RESPONSE TO TWODIFFERENT DIETARY PROTEIN SOURCES
IN NEWLYWEANED PIGLETS.
Caroline A. Makkink1, Pierre J.M. Berntsen1,
Brigitte ML. op den Kamp1, Bas Kemp1,2, Martin W.A. Verstegen1
1
2
Agricultural University, Department of Animal Nutrition,
Haagsteeg 4,6708PM Wageningen, TheNetherlands,
present address:Agricultural University, Department of Animal Husbandry,
Marijkeweg 40,6709PG Wageningen, The Netherlands
Submitted to Journal of Animal Science
85
Chapter V
Summary
Seventy piglets were weaned at 25 days of age and fed diets based on either skim-milk
powder (SMP)or soybean protein concentrate (SOY).At 0,3,6and 10daysafter weaning,
piglets were anaesthetized, their pancreas removed, and digesta collected from different
sections of the digestive tract. The ratio of trichloro acetic acid (TCA) precipitable protein
to total (crude) protein (pp/cp) in gastric digesta washigherwith SOY feed than with SMP
feed. In the jejunum no difference was found, hence, the degree of protein breakdown in
jejunal chymedidnot differ between protein sources.Trypsin activitiesinjejunal chymeand
in pancreatic tissue increased after weaning. Chymotrypsin activity in pancreatic tissue
tended to decrease after weaning and did not reach 'weaning-levels' for at least ten days.
Pancreatic trypsin developed more rapidly than chymotrypsin after weaning. Chymotrypsin
activities injejunal digesta washigher for the SMPfed piglets than for the SOY fed piglets.
Protease activities inthejejunum at day6after weaningwere clearlyaffected byfeed intake
after weaning.The ratio between trypsin and chymotrypsin activityin pancreatic tissue and
jejunal chyme was higher for SOY fed piglets than for SMP fed piglets. It was concluded
that the stomach plays an important role in the digestion of milk protein and that the
development of pancreatic proteases after weaning (synthesis, secretion, breakdown)
depends on feed intake and on dietary protein source.
Introduction
Newly weaned piglets often show depressed feed intake and growth when fed vegetable
proteins compared to proteins from animal sources.As the piglets get older, the digestion
ofvegetable protein increases,while the digestion of milkprotein ishigh immediately after
weaning and hardly increases with advancing age of the piglets (Decuypere et al, 1981,
Wilson & Leibholz, 1981a,c, Owsley et al, 1986a). Immediately following weaning, the
digestive system of the piglet has to adapt to the new feeding regimen with respect to pH,
enzyme secretion and gut motility.
Itiswellknown that soyprotein for example,can cause digestive problems inyoungpiglets,
due to the presence of antinutritional factors, oligosaccharides and/or antigens. Also, the
type of protein itself maycause digestive disturbances, especially inyoungpiglets (Giesting,
1986, Sissons, 1989).
After weaning, pancreatic enzyme activities may decline dramatically (Lindemann et al,
86
Chapter V
1986, Owsley et al, 1986b). The development of pancreatic proteases after weaning may
thus depend on the protein source in the diet as well as on the amount of feed ingested.
This experiment was undertaken to study the effects of skim-milk powder and soy protein
concentrate on feed intake, protein breakdown, digesta pH and trypsin and chymotrypsin
activity in digesta and pancreatic tissue of newly weaned piglets.
Materialsand Methods
Animals, housing, feeding
Ten litters of seven pigletseach (Great York*(Great York*Dutch Landrace))were weaned
at 25 days of age. No creep feed was provided during the suckling period. After weaning,
the piglets were housed individually in wooden crates (60*70 cm) with bedding. Visual
contact between pigletswasnot possible.Within litters,pigletswere allocated to one oftwo
dry, pelleted diets. Weaning weights were similar within litters, between diets. The
composition of the diets is given in Table 1. Diets were formulated to contain equal
amounts of protein, energy, fiber, fat and lactose. Skim-milk powder and soy protein
concentrate were the sole sources of protein in the SMP and the SOY diet, respectively.
The piglets were fed twice daily (morning and afternoon) at an 8 hour interval. From
weaning onwards, the morning feed was given one hour before the designated time of
sampling and the evening feed was given 8hours later. Daily feed allowance was restricted
to 5% of liveweight.
Sampling
At weaning (day 25), one piglet from each litter was taken from the sow, anaesthetized
through inhalation anaesthesia (N 2 0, 0 2 , halothane, no premedication) and sampled. At
3, 6and 10days after weaning (day 28,31and 35,respectively) two piglets from each litter
(one on the SMP diet and one on the SOY diet) were anaesthetized one hour after the
morning feeding. The piglets were weighed, and exsanguinated from thejugular veins and
artery. An off midline incision was made to expose the digestive tract. The stomach was
secured insitu and removed from the abdominal cavity.The small intestine was divided in
'duodenum' (first two meters of the small intestine from pylorus), 'ileum' (last two meters
of small intestine up to the ileocecal valve) and 'jejunum' (remaining middle section of the
small intestine) and removed from the abdominal cavity.The different parts were emptied
into aluminium foil trays. The contents were weighed, mixed and the pH measured. One
87
ChapterV
gramofdigesta (gastricandjejunal)weremixedwithtwomloftrichloro aceticacid solution
(TCA, 30 g/1) to precipitate the non-absorbable fraction of the nitrogenous material
(Ternouth et al, 1974, Souffrant et al, 1991). These digesta samples were kept in a
refrigerator (0-4°C)untilanalysis.The remainder ofthedigestawasfrozen immediatelyat20°C.
Table 1.
Composition of the diets(%).
SMP
Ingredients:
maize/wheat starch
skim-milk powder
soy protein concentrate (63%CP) 1
dextrose
lactose
soyaoil
cellulose
ground limestone
mono Caphosphate
salt
KHCO s
NaHCO s
L-lysine-HCl
DL-methionine
L-threonine
vit/min premix
Contents (calculated from ineredient comDositionl:
dry matter
crude protein
digestible crude protein
ether extract
crude fiber
crudeash
starch
net energy (MJ/kg)
Analyzed contents:
dry matter
crude protein
SOY
29.50
46.00
13.44
2.00
5.00
.75
.50
.30
.30
.10
.11
1.00
28.90
25.40
11.06
22.50
2.20
4.10
1.45
2.25
.30
.20
.20
.14
.22
.08
1.00
93.80
16.21
15.43
2.51
4.95
5.46
25.71
10.21
93.95
16.44
14.72
2.44
4.95
5.79
25.71
10.26
89.71
16.89
88.98
16.74
Soycomil-P: Loders Croklaan, Wormerveer, TheNetherlands. Trypsin Inhibitor Activity: less than 3mg inhibited
trypsin per gram (95%reduction compared tountoasted soybean meal). Urease: less than .1mg N/g*minute at 30
C. Antigens:notpresent.
88
Chapter V
The pancreas was excised, freed from connective tissue, weighed and frozen immediately
at -20 °C. The whole procedure took approximately 20 minutes per piglet.
The frozen digesta and pancreas of each piglet was freeze dried and ground within two
weeks of sampling. Samples for enzyme analysis were stored at -20 °C under N2 gas to
prevent enzyme activity loss.
Analyses
Gastric and jejunal digesta were analyzed for TCA-precipitable protein (Ternouth et al,
1974) and crude protein content. Jejunal digesta and pancreatic tissue samples were
analyzed for trypsin and chymotrypsin activity (Bergmeyer, 1974).Zymogens in pancreatic
tissue samples were activated with porcine enterokinase (Sigma Chemical) before enzyme
analyses.Trypsin and chymotrypsin activitiesweremeasured at pH 8.1and 7.8,respectively,
usingTAME(Na-toluolsulphonyl-L-arginine-methylester,Merck)andBTEE(N-benzoyl-Ltyrosine-ethylester, Fluka Biochemika) as respective substrates (Bergmeyer, 1974).
Extinction changes were measured at 247 and 256 nm for trypsin and chymotrypsin
respectively, using a spectrophotometer (Beckman DU-64).
An analysisofvariancewasperformed separatelyfor each dayofslaughter usingSAS-GLM
(SAS, 1990) according to the following model: YSj = /i + PSj + b / F I + e^
In which:
Y
i.i
M
PSi
bx*FI
C::
= dependent variable
= overall mean
= dietary protein source (i = 1, 2)
= feed intake, overall covariable
= error term
The effect offeed intakewithinprotein source(interaction between protein sourceand feed
intake) was found to be non-significant for any of the parameters and was therefore not
included from the model. To determine the effect of protein source on feed intake, the
following model was used: Yjj = n + PS{ + e^
In which:
Y;j
= dependent variable (feed intake)
H
= overall mean
PS; = dietary protein source (i = 1, 2)
etj
= error term
89
Chapter V
To reveal overall changes and trends during the post weaning period, the following model
was used:
Yy = n + SDj + e^
In which:
Yy
= dependent variable
H
= overall mean
SD; = day of sampling (i = 0, 3, 6, 10)
e^
= error term
Results
Feed intake, average daily gain, pancreatic weight and digesta pH
At weaning, the piglets were 25 days of age and weighed 7.15 kg (sd = 0.98, n = 70).
No diarrhoea or other signs of illness were observed throughout the experiment.
Feed intake and average daily gain are presented in Table 2. During the first three days
postweaning,no significant differences infeed intakeorgrowthwere found between protein
sources. Variation in feed intake and growth was considerable during this period. Feed
intake affected average daily gain during the whole post-weaning period (10 days).
From 3 days after weaning, differences in average daily gain between protein sources
(p<0.05) appeared. Piglets fed on SMP had a higher average daily gain from 3days after
weaning onwards compared to piglets on SOY. Feed intake affected average daily gain
during the whole post-weaning period (10 days).
The weight of the pancreas (absolute, and relative to liveweight) increased after weaning
(Figure 1).Feed intake and protein source did not affect theweight of the pancreas (Table
3).
No effects of feed intake or protein source on digesta pH were detected (p>0.05), except
for the pH in the ileum which was higher for the SOY fed piglets. Changes in
gastrointestinal pH are depicted in Figure 2.The pH in the gastrointestinal tract gradually
increased from the proximal to the distal section of the small intestine.
90
Chapter V
Table 2.
Feed intake (FI, gram per day) and average daily gain (ADG, gram per day) per
post-weaning period (LSMeans, b=coefficient of regression).
feed intake
b
p
proteinsource
SMP
SOY
P
FI (g/d):
day 0-3
day 0-6
day 0-10
173
215
256
178
234
234
.8843
.5825
.3561
ADG (g/d):
day 0-3
day 0-6
day 0-10
5
157
185
47
63
131
.5551
.0072
.0193
1
2
3
4
rootMSE:
83
78
52
.02 .9571
.40 .0679
.74 .0025
5
6
7
8
9
155
68
46
10
days after weaning
Figure 1.
Pancreatic weight (fresh tissue), absolute (g) and relative to liveweight (g/kg),
LSMeans with StdErr of LSM.
91
Chapter V
10
6 "
*bc
-©—
duodenum
J
0
1
2
'
3
— £ —
i
4
Jtjunum
I
5
I
6
I
7
8
a
(l«um
I
1
9
10
days after weaning
Figure 2.
Digesta pH indifferent sectionsof the digestive tract.LSMeans with StdErr of LSM.
1500
-*-
Ptfyp
T
Pehym
J*
C
1000
yTb
1/
500
---•a
—
i
* "
'
0
1
'
2
i
3
1
i
4
5
1
6
1
7
I
8
i
9
i
10
days after weaning
Figure 3.
Trypsin andchymotrypsin activities inpancreatic tissue(Units pergram freeze dried
tissue). LSMeans with StdErr of LSM.
92
Chapter V
Protein breakdown in stomach and jejunum
The source of dietary protein clearly affected (p<0.01) the degree of protein breakdown
in the stomach (Table 4).The ratio precipitable protein / crude protein (pp/cp) was lower
for the SMP-diet, indicating a greater degree of protein breakdown. In the diets, this ratio
was 0.79 and 0.82 for the SMP and the SOY diet, respectively. Feed intake did not affect
the pp/cp ratio in the stomach. The ratio precipitable protein / crude protein in the jejunal
digesta, was not affected by protein source nor feed intake.
Enzyme activities in pancreas and jejunum
Protease activities in pancreatic tissue and jejunal digesta are shown in Table 4. Trypsin
activity in pancreatic tissue (units per gram freeze dried tissue) increased after weaning
(Figure 3). No effects of feed intake or protein sources on pancreatic protease activities
(U/g freeze dried material) were found (p>0.05). At ten days after weaning, the
chymotrypsin activities in pancreatic tissue for the SMP groups were similar to the
chymotrypsin activities at the day of weaning.
Trypsin activities injejunal digesta (units pergram freeze dried digesta) increased markedly
after weaning. At ten days after weaning, trypsin activity in the jejunal digesta was 7 to 8
times as high as the activity at the dayof weaning.Chymotrypsin activity injejunal digesta
also increased after weaning, but at a much slower rate than trypsin activity. At 6 and 10
daysafter weaning,pigletsfed theSMPdiethad higherchymotrypsin activitiesinthejejunal
digesta than piglets fed the SOY diet (p<0.05).
At 6daysafter weaning,jejunal protease activities(per gram freeze dried material and total
units) were affected by feed intake. This was not found on day 3 or day 10after weaning.
The total amount of trypsin and chymotrypsin in pancreas and jejunum at day 6 was
positively influenced by feed intake.
The ratio between trypsin and chymotrypsin activity in pancreatic tissue and in jejunal
digesta are given in Table 4.At 6and 10days after weaning, the ratio between trypsin and
chymotrypsin activity injejunal digesta was higher for the SOY fed piglets.This effect was
not found in the pancreas. Feed intake did not affect the tryp/chym ratio.
93
ChapterV
Table 3.
Liveweights (g), pancreas weights (g), digesta weights (g) and pH in the digestive
tract(LSMeans, b=coefficient of regression). LW=liveweight, PW=pancreasweight,
relPW-pancreas weight relative to liveweight (g/kg).
at weaning:
day 0
protein source
LW(g)
PW(g)
relPW(g/kg)
digestaweights:
stomach (g)
duodenum (g)
jejunum (g)
ileum (g)
total (g)
digestapH:
stomach
duodenum
jejunum
ileum
-
after weaning:
day 3
SMP SOY p
7016
10.4
1.47
7303
9.25
1.28
7333
9.47
1.29
feedintake:
.9501
.7678
.8626
b
P
2.63
-.00
-.00
.3876
.6893
.1309
.23
.01
.30
.3155
.3083
.2067
.8537
.2230
80
3
41
3
127
132
4
126
17
279
170
5
139
19
333
.2936
.5079
.7345
.7561
.4294
3.07
4.67
6.45
7.13
3.83
5.41
6.48
7.05
4.03
5.23
6.52
6.97
.7472
.5785
.7640
.7129
SOY
P
b
P
.2754
.1154
.2669
10.82
0.03
0.00
.0014
.0022
.1175
.26
.05
.26
.1649
.0850
.7664
.1567
.2930
.00
.00
.00
.00
.4121
.5493
.3324
.2872
-.00
.54
.01
-.01
.00
.00
.1192
.0441
.6769
.6736
day6
protein source
LW(g)
PW(g)
relPW (g/kg)
digesta weights:
stomach (g)
duodenum (g)
jejunum (g)
ileum (g)
total (g)
digestapH:
stomach
duodenum
jejunum
ileum
SMP
8048 7570
13.20 11.12
1.47
1.63
222
6
187
21
435
241
7
142
15
404
.4997
.3640
.0435
.2671
.3905
4.81
5.26
6.44
6.90
4.86
5.19
6.56
7.30
.8832
.8548
.3949
.0071
SOY
-.02
-.04
day10
protein source
LW(g)
PW(g)
relPW (g/kg)
digesta weights:
stomach (g)
duodenum (g)
jejunum (g)
ileum (g)
total(g)
digestapH:
stomach
duodenum
jejunum
ileum
P
b
8744 8665
13.80 13.42
1.54
1.58
.8525
.7025
.7208
17.66
237
3
172
13
425
253
13
206
17
489
.7431
.0063
.4612
.4756
.2812
1.01
4.15
4.44
6.36
6.95
4.09
5.07
6.45
6.46
.9393
.3579
.5896
.4869
.00
.00
SMP
94
P
.04
.00
.06
.69
.05
1.81
-.00
-.00
.0005
.0011
.1939
.0472
.0605
.1402
.4269
.0053
.6865
.5443
.6939
.5677
Chapter V
Table 4.
Ratio precipitable protein to crude protein (pp/cp) and enzyme activities in the
digestive tract (LSMeans). (P=pancreas tissue; J=jejunum digesta; tryp=trypsin
activity,unitspergramfreeze dried material;chym=chymotrypsin activity,units per
gram freeze dried material; tot=total activity in pancreas or jejunum).
protein source
ratio pp/cp:
stomach
jejunum
enzyme activities:
Ptryp (U/g)
Pchym (U/g)
Jtryp (U/g)
Jchym (U/g)
Ptryptot (Units)
Pchymtot (Units)
Jtryptot (Units)
Jchymtot (Units)
total trypsin (P+J)
total chym. (P+J)
Ptryp/Pchym
Jtryp/Jchym
at weaning:
day 0
after weaning
day 3
_
SMP
SOY
P
.67
.49
.58
.95
.99
.85
.0025
.6158
-.00
-.00
.4367
.7725
404
371
55
51
1028
943
244
192
1358
1246
1.17
1.77
612
271
243
92
1237
561
1521
502
2758
1062
2.74
2.83
452
194
194
60
919
385
1478
419
2398
805
3.18
3.69
.3178
.4876
.5474
.0998
.3382
.4601
.9230
.5091
.4483
.2245
.4838
.3681
.06
-.33
-.71
-.19
-.27
-.85
-.16
.07
-.43
-.78
-.00
-.00
.9540
.6415
.1744
.1279
.8956
.5679
.9547
.9275
.8853
.5532
.8509
.7525
SMP
SOY
P
-
.56
.46
.86
.67
.0011
.0966
- .00
- .00
.1815
.8864
-
808
236
345
94
2130
667
5018
1441
7148
2108
4.16
3.82
979
208
378
73
2382
499
3741
688
6123
1187
4.90
5.27
.4805
.6207
.4474
.0291
.6883
.3392
.0887
.0032
.3413
.0106
.5599
.0255
.29
- .02
.90
.13
4.74
1.06
20.21
4.43
24.95
5.49
.00
.00
.8548
.9534
.0051
.0462
.2664
.3660
.0005
.0078
.0023
.0201
.7337
.2915
feed intake:
day 6
protein source
ratio pp/cp:
stomach
jejunum
enzyme activities:
Ptryp (U/g)
Pchym (U/g)
Jtryp (U/g)
Jchym (U/g)
Ptryptot (Units)
Pchymtot (Units)
Jtryptot (Units)
Jchymtot (Units)
total trypsin (P+J)
total chym. (P+J)
Ptryp/Pchym
Jtryp/Jchym
(continued)
95
~f
Chapter V
Table 4.
(continued)
protein source
ratio pp/cp:
stomach
jejunum
enzyme activities:
Ptryp (U/g)
Pchym (U/g)
Jtryp (U/g)
Jchym (U/g)
Ptryptot (Units)
Pchymtot (Units)
Jtryptot (Units)
Jchymtot (Units)
total trypsin (P+J)
total chym. (P+J)
Ptryp/Pchym
Jtryp/Jchym
at weaning:
after weaning:
day 10
_
SMP
SOY
P
b
P
-
.50
.69
.91
.62
.0047
.7944
- .00
.00
.2516
.1042
-
1247
308
399
90
3664
926
4998
1154
8661
2080
4.37
4.55
1225
234
424
59
3280
633
6040
846
9320
1479
5.69
7.72
.9145
.1785
.5859
.0246
.5658
.0985
.3841
.2435
.6019
.0723
.1428
.0002
-1.71
.06
.14
.06
4.13
2.39
21.72
3.56
25.86
5.95
- .00
.00
.4164
.9106
.7607
.6567
.5332
.1683
.0780
.1773
.0509
.0727
.6346
.6800
feed intake:
Discussion
Feed intake, average daily gain, pancreatic weight and digesta pH
The high variation in feed intake as noticed in the present experiment is a phenomenon
often observed in practical piglet feeding and also known from literature (Armstrong and
Clawson, 1980 and Cranwell and Moughan, 1989). The higher average daily gain on the
SMP diet, also, is in accordance with findings from literature (Walker et al, 1986).
The relative weight of the pancreas increased especially between day 3 and day 6. This
positive allometryis in accordance with data from Owsleyetal. (1986b) and Lindemann et
al. (1986).
The pH of digesta increased along the digestive tract due to the addition of more alkaline
substances to the digesta (pancreaticjuice,bile,gutwall secretions).After weaning, the pH
in the stomach was high (3.8-4.9) compared to the optimal pH for pepsin action (pH 2-4,
Beynon and Bond, 1989),whilepH in the smallintestinewaslow(4.4-7.3) compared to the
optimum pH for trypsin and chymotrypsin action (pH 7.8-8.1, Beynon and Bond, 1989).
However, the pH wasmeasured in the total, mixed chyme of each segment, soit is possible
that at some sites in the stomach (e.g. close to the gastric wall or at the pyloric site, as
reported by Kamphues (1987)) pH was lower than the values reported in Table 3.
96
Chapter V
The pH values agreewith the data of Newport and Keal (1982),Schnabel etal.(1982) and
Wilson and Leibholz (1981b).
Protein breakdown in stomach and jejunum
TheTCA-precipitableprotein fraction indigesta canberegarded asnot directly absorbable
(Souffrant, 1991),since it consists of the larger nitrogen containing molecules such as nondigested feed protein and enzymes.The ratio between TCA-precipitable protein and crude
protein (pp/cp ratio) isan indication for the degree ofdietary protein breakdown, especially
in the stomach. In the stomach a relatively small proportion of the N compounds consists
of secreted enzymes and mucus.
A clear difference was found in gastric precipitable protein /crude protein ratio (pp/cp)
between SMP fed and SOY fed piglets, indicating a difference in degree of protein
breakdown between the two diets. This difference probably cannot be attributed to
antinutritional factors or antigens, since levels of these are very low in the soy protein
concentrate used in this study (Table 1). It may be hypothesized that the different gastric
pp/cp ratio may have consequencies for further breakdown and thus absorption of
hydrolyzed protein invarious parts of the gastrointestinal tract. It appears that the stomach
could play an important role in the difference in protein digestibility between soy and milk
protein. The clotting capacity of milk proteins may play a major role in this respect. As a
result, the emptying of the stomach might also differ between the two protein sources as
was found by Zebrowska et al. (1983) and by Laplace et al. (1984). Unfortunately, gastric
emptying could not be measured in this experiment. The pp/cp ratio could be a useful
parameter in the study of the digestion (breakdown) of protein in the stomach where
absorption is probably negligible or zero (Bayley, 1978). Feed intake had no effect on the
pp/cp ratio in the stomach.The amount of feed consumed at the last mealbefore slaughter
might have had an influence on the degree of protein breakdown. The amount of gastric
digesta at slaughter (asan indicator of the last meal size)wasaffected byfeed intake at day
10 (Table 3).
At the jejunal level, no effect of protein source on pp/cp ratio was found. This is in
agreement with results from Newport and Keal (1982),whoweaned piglets at 2daysof age
onto liquid diets containing either skim-milk powder, soybean isolate or soybean
concentrate.
Enzyme activities in pancreas and jejunum
Although trypsin and chymotrypsin activities in pancreatic tissue andjejunal digesta (Units
per gram freeze dried material) were variable between piglets, a systematic increase in
97
Chapter V
trypsin activityinpancreas andjejunum after weaningwasfound. Incontrastwithdata from
Lindemann et al. (1986) and Owsley et al. (1986b), no post-weaning decline in trypsin
activity was found, which could be due to differences in breed or age of piglets or feed
intake.At day6,a significant positiveeffect offeed intakeonjejunal protease activities (per
gram freeze dried digesta and total units) was found. Therefore, it can probably be
concluded that the decline in enzyme activities found by Lindemann et al. (1986) and
Owsley et al. (1986b) is related to low feed intake after weaning rather than to weaning
itself. Chymotrypsin activity per gram freeze dried pancreatic tissue did diminish after
weaning, whereas chymotrypsin activity per gram freeze dried jejunal chyme increased.
Total chymotrypsin activity in the pancreas and in the jejunum showed the same
development after weaning. From these data it appears that chymotrypsin synthesis cannot
keep up with chymotrypsin secretion or that the rate of hydrolysis of chymotrypsin in the
digestive tract changes after weaning.
No effect of dietary protein source on trypsin or chymotrypsin in pancreatic tissue was
found. Newport and Keal (1982) did find higher trypsin and chymotrypsin activities in
pancreatic tissues of soy protein concentrate fed piglets. These conflicting results are
probably due to differences in weaning age (2 days vs 25 days) and post weaning diets
(liquid vs dry diets, dietary crude protein content 24 vs 16%, other type of soy protein
concentrate).
Variation infeed intake during the first three daysafter weaningwasnot related to enzyme
activities at day 3. Most regression coefficients on feed intake were negative at this time,
indicating limitations of the enzyme system during the early post-weaning period. Thiswas
also suggested by Lindemann et al. (1986).
At day 6, feed intake between weaning and sampling clearly had positively affected jejunal
trypsin and chymotrypsin activities.The suggestion that post-weaning feed intake could be
a major factor in pancreatic enzyme synthesis and secretion has previously been made by
Owsley et al. (1986b). At day 10, the effect of feed intake on enzyme activities was still
positive, but not significant anymore.
The fact that, at day 6, not only total protease activities injejunal digesta but also protease
activities per gram freeze driedjejunal digesta increased with increasing post-weaning feed
intake suggests that, at this time,pancreatic protease synthesis and secretion isnot yet fully
adapted to the changed feed intake after weaning. This is also illustrated by the increase
of jejunal trypsin activity (units per gram freezedried digesta) between day 0 and day 6.
After day 6,this activity remains fairly constant at approximately 400units per gram freeze
dried digesta.
With SMP fed piglets,jejunal chymotrypsin activity was higher than with SOY fed piglets.
98
Chapter V
This effect has also been reported by Owsley et al. (1986b) and by Newport and Keal
(1982).
The difference injejunal trypsin/chymotrypsin ratio between SMP and SOY at day 6 and
10 indicates an effect of dietary protein source on specific protease (either trypsin or
chymotrypsin) synthesis and secretion and/or a diet-dependent breakdown of pancreatic
proteases in the gastrointestinal tract.
From the changes in trypsin/chymotrypsin ratio (t/c ratio) in pancreatic tissue and jejunal
digesta it isconcluded that trypsin and chymotrypsin synthesis, secretion and/or breakdown
is regulated independently as previously been suggested by Efird et al. (1982) in pigs and
bySnook and Meyer (1964)inrats.Independent regulation ofsynthesis/secretion might also
be expected from the specificity of thetwoenzymes:trypsin preferably cleavespeptic bonds
next to the alkaline amino acids arginine or lysine.Chymotrypsin islessspecific and cleaves
peptic bonds involving phenylalanine, tryptophane, tyrosine (aromatic amino acids) or
leucine (Wood et al, 1974).
Arginine, particularly is more abundant in the soy protein concentrate used in this
experiment compared to the skim-milk powder (7.2 vs 3.0 g/100 g of amino acids,
respectively). From this it isexpected that the t/cratiowillbe higher in piglets fed the SOY
diet.
From this experiment it can be concluded that the stomach probably plays a major role in
the digestion of milk protein. According to Pekas et al. (1964), soy protein digestion
depends more on pancreatic digestion. However, SOY fed piglets did not show higher
pancreatic enzyme activities in pancreatic tissue or jejunal chyme than SMP fed piglets.
Therefore, itcan be concluded either that SMP and SOY are equallywell utilized bynewly
weaned piglets or that differences in protein digestibility between milk proteins and soya
proteins are caused by differences in gastric protein digestion, absorption and/or by
differences in endogenous nitrogen secretion. The latter has been found to be higher with
soyproteins compared tomilkproteins (Makkink etal, unpublished results).The pancreas
is probably not the major source of this increase in endogenous nitrogen, since the activity
of two major nitrogen containing components (trypsin and chymotrypsin) did not differ
between the two protein sources. The first three days after weaning comprise a difficult
period for the young pig: during this time, pancreatic enzyme activities in pancreatic tissue
and jejunal digesta do not respond to dietary protein source or feed intake.
99
Chapter V
Implications
Early weaned piglets encounter several problems during the process of adaptation to post
weaning solid feeds. Major digestive processes such as feed intake patterns, enzyme
synthesis and secretion, motility and absorption need to be modified to meet the changing
demands imposed onthe piglet's digestivetract. It isunlikely,therefore, that onlyone small
part in this intricate system could be the sole source of postweaning problems in the pig.
Especially the often lowand irregular feed intake of newlyweaned piglets mayhamper the
development and adaptation of the digestive capacity in these animals. Improving the
piglet's feed intake (total amount and frequency of meals) during the early post-weaning
period could stimulate the development and adaptation of the digestive system. However,
thefirst three post-weaning dayswillprobablyremain strenuous for the earlyweaned piglet.
References
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Nutrition and management of early weaned pigs: Effect of increased nutrient
concentrations and(or) supplemental liquid feeding./. Anim. Sci.50(3):377-383
Bayley, H.S., 1978.
Comparative physiology of the hindgut and its nutritional significance.J. Anim. Sci.
46:1800Bergmeyer, H.U. 1974.
Methoden der Enzymatischen Analyse. ThirdEdition. Weinheim: Verlag Chemie.
Beynon, R.J., Bond, J.S. (editors). 1989.
Proteolytic Enzymes, a Practical Approach. PracticalApproach Series. IRL Press at
Oxford University Press,Oxford, UK
Cranwell, P.D., Moughan, P.J., 1989.
Biological limitations imposed bythe digestive system to the growth performance of
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Decuypere, J.A., Meeusen, A., Henderickx, H.K., 1981.
Influence of the partial replacement ofmilkprotein bysoybean protein isolateswith
different physical properties on the performance and nitrogen digestibility of earlyweaned pigs./. Anim. Sci. 53(4):1011-1018
100
Chapter V
Efird, R.C., Armstrong, W.D, Herman, D.L., 1982.
The development of digestive capacityinyoungpigs:effects ofweaningregimen and
dietary treatment. /. Anim. Sci. 55(6):1370-1379
Giesting, D.W., 1987.
Utilization of Soy Protein by the Young Pig. Thesis. University of Illinois,UrbanaChampaign, USA.
Kamphues, J., 1987.
Untersuchungen zu Verdauungsvorgangen bei Absetzferkeln in Abhangigkeit von
Futtermenge und -zubereitung sowie von Futterzusatzen. Thesis. Tierarztliche
Hochschule Hannover, Hannover, Germany.
Laplace, J.P., Pons, O., Cuber, J.C., Kabore, C , 1984.
Evacuation gastrique et composition en acides amines desproteines du regime chez
le pore.Ann. Zootech.33:59Lindemann, M.D., Cornelius, S.G., El Kandelgy, S.M., Moser, R.L., Pettigrew, J.E., 1986.
Effect of age,weaning and diet on digestive enzyme levelsinthe piglet./ Anim. Sci.
62(5):1298-1307
Newport, M.J., Keal, H.D., 1982.
Artificial rearing of pigs. 12.Effect of replacement of dried skim-milk by either a
soya-protein isolate or concentrate on the performance of the pigs and digestion of
protein. Br.J.Nutr. 48(l):89-96
Owsley, W.F., Orr, D.E., Tribble, L.F., 1986a.
Effects of nitrogen and energy source on nutrient digestibility in the young pig. /.
Anim. Sci. 63:492-496
Owsley, W.F., Orr, D.E., Tribble, L.F., 1986b.
Effects of age and diet on the development of the pancreas and the synthesis and
secretion of pancreatic enzymes in the young pig./ Anim. Sci. 63:497-504
Pekas, J.C., Hays, V.W., Thompson, A.M., 1964.
Exclusion of the exocrine pancreatic secretion: effect on digestibility of soybean and
milk protein by baby pigs at various ages./. Nutr. 82:277-286
SAS Institute Inc., 1990.
SASProcedures Guide.Version6, ThirdEdition. Cary, NC:SAS InstituteInc., 705pp.
Schnabel, E., Schneider, R., Schubert, C, 1982.
Untersuchungen zum pH-Wert im vorderen Trakt beim Absetzferkel. Arch.
Tieremahrung 32(9):631-635
101
Chapter V
Sissons, J.W., 1989.
Aetiology of diarrhoea in pigs and pre-ruminant calves. In: Recent Advances in
Animal Nutrition. Eds. W.Haresign, DJ~A. Cole.Butterworths, London, UK
Souffrant, W.-B.,1991.
Endogenous nitrogen losses during digestion in pigs. In: Proceedingsof the Vth
International Symposium on Digestive Physiology in Pigs,Wageningen (Doorwerth),
Netherlands, 24-26 April 1991 (EAAP Publication No. 54, 1991). Eds. M.W^A.
Verstegen, J.Huisman, L~A. den Hartog. pp.147-166.
Snook, J.T., Meyer, J.H., 1964.
Response of digestive enzymes to dietary protein.J.Nutr.82:409-414
Ternouth, J.H., Roy, J.H.B., Siddons, R.C., 1974.
Concurrent studies of the flow of digesta in the duodenum and of exocrine
pancreatic secretion of calves. 2. The effects of addition of fat to skim-milk and of
'severe' preheating treatment of spray-dried skim-milk powder.Br. J.Nutr. 31:13-26
Walker, W.R., Maxwell, C.V., Owens, F.N., Buchanan, D.S., 1986.
Milk versus soybean protein sources for pigs: 1. Effects on performance and
digestibility./ Anim. Sci.63(2):505-512
Wilson, R.H., Leibholz, J., 1981a.
Digestion in the pigbetween 7and 35days of age. 1.The performance of pigsgiven
milk and soyabean proteins. Br.J.Nutr. 45:301-320
Wilson, R.H., Leibholz, J., 1981b.
Digestion in the pig between 7 and 35 days of age. 2.The digestion of dry matter
and the pH of digesta in pigs given milk and soyabean proteins.Br. J.Nutr. 45:321336
Wilson, R.H., Leibholz, J.. 1981c.
Digestion in the pig between 7and 35 days of age. 3.The digestion of nitrogen in
pigs given milk and soyabean protein. Br.J.Nutr. 45:337-346
Wood, W.B., Wilson, J.H., Benbow, R.M., Hood, L.E. (editors). 1974.
Biochemistry, a Problems Approach. TheBenjamin/Cummings PublishingCompany,
Menlo Park,California,USA.
Zebrowska, T., Low, A.G., Zebrowska, H., 1983.
Studies on gastric digestion of protein and carbohydrate, gastric secretion and
exocrine pancreatic secretion in the growing pig.Br.J.Nutr. 49:401-410
102
ChapterVI
EFFECT OF DIETARY PROTEIN SOURCE ON FEED INTAKE, GROWTH,
PANCREATIC ENZYMEACTIVITIES AND JEJUNAL MORPHOLOGY
IN NEWLYWEANED PIGLETS.
Caroline A. Makkink1, George Puia Negulescu1,2,
Qin Guixin1,3, Martin W.A. Verstegen1
1
Agricultural University, Department of Animal Nutrition, Haagsteeg4,
6708PM Wageningen, The Netherlands
Ministerul Agriculturii, Academia deStiinte Agricole siSilvice Directia Generala
Zooveterii, Institutul de BiologiesiNutritie Animala,
8113Balotestisector Agricol Ilfov, Roumania
3
Jilin Agricultural University, Department of Animal Science,Jingyue,Changchun,
130118,China
Submitted to British Journal of Nutrition
103
Chapter VI
Summary
Seventy piglets with no access to creep feed were weaned at 28 days of age and fed
one of four diets based on either skim milk powder (SMP), soya protein concentrate
(SPC), soyabean meal (SBM) or fish meal (FM). At 0, 3, 6 and 10 days after
weaning, piglets were euthanized and the pancreas and digesta from stomach and
small intestine were collected, freeze-dried and analyzed for dry matter (DM)
nitrogen (N) and trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) activity. Small
intestinal tissue samples were taken to judge gut wall morphology. Results indicated
that dietary protein source affected post-weaning feed intake, pancreatic weight,
gastric pH and gastric protein breakdown, and pancreatic and jejunal trypsin and
chymotrypsin activity. Post-weaning feed intake appeared to be an important factor in
digestive development of newly weaned piglets.
Introduction
In pigs, digestive disorders are frequently encountered in the early post-weaning
period. The adaptation of the gastrointestinal tract with regard to motility, secretion,
digestion and absorption is often impaired, resulting in poor feed intake, growth
(Okai et al, 1976), and health status. These problems are at least partly related to
the transition from sows milk to solid post-weaning diets containing different protein
and energy sources.
After birth, the protein digestive capacity of young piglets is adapted to the digestion
of milk proteins. Proteins of plant origin are digested to a lesser extent than milk
proteins resulting in poor performance when fed to newly weaned piglets (Wilson &
Leibholz, 1981a, 1981c).
This experiment was designed to study the development of pancreatic enzymes of
newly weaned piglets in relation to dietary protein source and post-weaning feed
intake.
Materialsand Methods
Seventy piglets with no access to creep feed during the suckling period were weaned
at 28 days of age. Ten piglets were anaesthetized immediately after weaning (day 0),
weighed and exsanguinated from the jugular veins and artery. The gastrointestinal
104
Chapter VI
tract was divided in four segments: stomach, duodenum (= first 2 meters from
pylorus), ileum (=last 2 meters of the small intestine) and jejunum (=remaining part
of the small intestine). Digesta were collected quantitatively from stomach and small
intestinal segments. In fresh gastric digesta trichloro-acetic acid (TCA) precipitable
protein was measured according to Ternouth et al (1974). Digesta samples were
weighed, pH measured and samples were frozen and stored at -20 °C until freezedrying. The pancreas was excised, freed from adhering tissues, frozen and stored at 20 °C until freeze-drying. After freeze-drying, stomach and jejunum digesta samples
were ground (1 mm) and analyzed for dry matter and crude protein content.
The TCA-precipitable protein fraction of the digesta comprises proteins and large
peptides which cannot be absorbed by the intestinal wall without further hydrolysis
(Souffrant, 1991).
The ratio between TCA-precipitable protein (pp) and crude protein (cp) in gastric
digesta was therefore calculated to determine the degree of gastric protein
breakdown.
Trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) activities were measured in
jejunal digesta and in pancreatic tissue after freeze-drying (Bergmeyer, 1974, Van
Baak et al., 1991).
The remaining sixty piglets were fed one of four experimental diets based on either
skimmilk powder (SMP), soya protein concentrate (SPC), soyabean meal (SBM) or
fish meal (FM). Diet compositions are given in Table 1. At 3, 6 and 10 days after
weaning, 5 piglets per diet were anaesthetized one hour after feeding 100 grams of
the diet. Samples were collected, processed and analyzed as described above (20
piglets per day). At 6 days after weaning, duplicate tissue samples (approximately
0.5cm x 0.5cm) were taken from the proximal and distal jejunum to assess villus
lengths and crypt depths. These tissue samples were stored in 3 ml cryotubes (Sanbio
BV Biological Products, P.O. Box 540, NL-5400 AM, Uden), frozen immediately in
liquid nitrogen and kept at -70 °C until further analysis. Sections were cut using a
cryostat (2800 Frigocut N, Reichert-Jung), stained with toluedine-blue and the length
of ten villi and the depth of ten crypts was measured in each sample.
Analysis of variance was performed using the GLM-procedure of SAS (SAS, 1990)
for each day of sampling separately using the following model:
Y„
=
/i + D, + bx*FI + e.u
in which Yy = dependent variable n = overall mean, D( = effect of diet (i = 1, 2,3,
4), bj*FI = effect of feed intake (overall co-variable) and e^ = error term.
105
Chapter VI
Composition of experimental diets
Table 1.
diet:
ineredient (%)
skimmilk powder1
soya protein concentrate2
soyabean meal 3
fish meal4
maize/wheat-starch
dextrose
lactose
soya oil
cellulose
ground limestone
mono Ca phosphate
NaCl
KHCO3
NaHCO,
L-lysine HC1
DL-methionine
L-threonine
vitamin and mineral premix
Cr 2 0 3
SMP
SPC
SBM
FM
47.00
-
-
25.40
-
-
34.40
-
21.30
34.17
10.96
22.50
2.00
5.00
0.75
0.25
0.30
1.10
0.40
20.78
10.96
22.50
2.75
2.85
1.45
2.10
0.30
1.00
0.10
28.90
10.96
22.50
2.20
4.10
1.45
2.25
0.30
0.20
0.20
0.14
0.22
0.08
1.00
0.10
calculated contents (%)
dry matter
crude protein (N*6.25)
ether extract
crude fiber
inorganic matter
net energy (MJ/kg)
93.80
16.21
2.51
4.95
5.46
10.20
93.95
16.44
2.44
4.95
5.79
10.25
93.30
16.38
3.18
5.02
5.99
10.21
93.88
16.22
3.54
4.95
5.05
10.49
analyzed contents (%)
dry matter
crude protein (N*6.25)
91.21
16.39
92.41
16.97
92.70
14.55
92.53
14.84
buffering capacity5
pellet hardness6
7.2
14.25
6.4
18.00
6.4
3.88
7.7
4.38
5
°
29.50
13.34
-
2.00
5.00
0.75
0.50
0.30
0.30
0.10
0.11
-
0.40
0.16
0.21
0.04
1.00
0.10
0.10
0.07
1.00
0.10
Crude protein 35.1 96
Trypsin Inhibitor Activity <0.5 mg inhibited trypsin per gram product. Antigens 2
titre log2. Protein Dispersability Index 3.93.
Soycomil P, Loders Croklaan BV, P.O.
Box 4, 1520 AA Wormerveer, The Netherlands
Crude protein 63.9 % Trypsin Inhibitor Activity 1.3 mg inhibited trypsin per gram jroduct. Antigens less
than 1titre log2. Protein Dispersability
Index 0.04.
Crude protein 39.2 %. Trypsin Inhibitoi Activity 1.6 mg inhibited trypsin per gram product. Antigens 5 titre
log2. Protein Dispersability Index 0.12.
Crude protein 69.2 %. Trypsin Inhibitor Activity 1.1 mg inhibited
trypsin per gram product. Antigens 2 titre
log2. Protein Dispersability Index 0.10.
ml 1N HC1needed to reach pH 4.00 in
*suspension of 20 g feed in 100 ml demineralieed water.
Determined by Kahl Pellet Tester (Amandus Kahl Nachf. Maschinenfabrik, 2057 Reinbek, Hamburg). Pellet
hardness expressed in kgf.
106
Chapter VI
Initially, the effect of feed intake within diet was also tested, but this was found to be
not significant for all variables except for chymotrypsin activity three days after
weaning and was therefore eliminated from the model.
Since the overall effect of feed intake on chymotrypsin activity on day 3 was not
significant, the effects of diet and of feed intake within diet on chymotrypsin activities
at day 3were analyzed according to the following model:
Y,
=
M+
D>+ b ^ F I + e ;j
in which Yy = dependent variable (chymotrypsin activity at day 3), n = overall
mean, D; = effect of diet (i = 1, 2, 3, 4), bj^FI = effect of feed intake within diet
and ey = error term.
To evaluate the development of gastrointestinal tissue weights after weaning the
following model was used:
Yij.
=
ur*+ W. +i e-
ij
in which Yy = dependent variable (gastric or intestinal tissue weight), Wj = day after
weaning (i = 0, 3, 6, 10) and e^ = error term.
Results
Feed intake and growth
At weaning piglets were 28 days of age and had a mean live weight of 7.1 kg
(SD=1.3, n=70).
Feed intake during the first three days post-weaning was affected by dietary protein
source (Table 2). Piglets on SBM and FM consumed more feed than piglets on the
SMP diet. Ten piglets out of 60 (5 on SMP, 3 on SPC and 2 on SBM) consumed less
than 50 grams of feed during the first three days after weaning. From three days
after weaning, feed intake was similar for all diets. Average daily gain was not
affected by dietary protein source (Table 2). During the first three days after
weaning, growth was very variable between piglets and feed intake strongly affected
average daily gain. From three days after weaning, no effect of feed intake on growth
was found (Table 2).
107
Chapter VI
Tissue weights
Dietary protein source did not influence the weight of the stomach and the small
intestine (absolute and relative to live weight) after weaning (Table 3a). The relative
weight of the small intestinal tissue decreased during three days after weaning
(Figure 1). The relative weight of gastric tissue increased gradually after weaning
(Figure 1).
2
4
6
8
days after weaning
SI
stomach
Figure 1.
Development of relative gastrointestinal tissue weights (gram per kg live
weight) after weaning (Least Square Means + Standard Error of LSM). Data
points within a tissue carrying different letters are significantly different
(p<0.05).
Pancreatic weight was clearly affected by dietary protein source. At day 3, the weight
of the pancreas (g per kg live weight) was higher for piglets fed SPC and FM than
for piglets fed SBM. At day 6, the pancreatic weight (g) was high for piglets fed SPC
and SMP and low for piglets fed FM. At day 10, the relative weight of the pancreas
was high for piglets fed SPC and low for piglets on the FM diet (Table 3a). At days 6
and 10 post-weaning, small intestinal tissue weight was positively affected by
preceding feed intake. At day 6 the same was found for the weight of the pancreas
(absolute and relative).
108
ChapterVI
Table2.
Effect of diet on average daily feed intake (FI) (Least Square Means) and
growth (ADG) (LSMeans(StdErr of LSM)).b • linear regression coefficient, p
= probability. LSMeans within a column carrying different superscripts are
significantly different (p<0.05)
FI(g/d)
number of piglets
day 0 - 3
60
day 3 - 6
40
day 6 - 1 0
20
SMP
SPC
SBM
FM
56 s
80 ab
95 b
112b
286
318
314
339
354
406
376
453
14
23
39
StdErr
ADG (g/d)
number of piglets
day 0 - 3
60
day 3 - 6
40
day 6 - 1 0
20
SMP
SPC
SBM
FM
26.0 (27.6)
- 6.3 (26.5)
- 0.4 (26.6)
25.7 (27.3)
224.3 (45.0)
292.8 (44.3)
215.4 (44.3)
273.0 (47.5)
268.2 (45.7)
256.7 (44.6)
172.5 (44.2)
217.4(52.5)
effect of feed intake:
linear regression coefficient
p (reg.coeff. = 0)
2.56
0.0001
0.27
0.3962
0.43
0.1598
Digesta pH
At days 3 and 6 after weaning, digesta pH was not affected by dietary protein source.
At day 3, jejunum pH was positively related to feed intake (Table 3b). At day 10,
piglets fed the soya diets had lower gastric pH than piglets fed the FM diet.
Gastric pp/cp ratio
The pp/cp ratio in the stomach digesta was lower for piglets fed the FM diet than for
piglets on SPC, indicating a higher degree of gastric protein breakdown in gastric
digesta present at the time of slaughter with piglets fed the FM diet (Table 3b).
Gut wall morphology
All piglets killed at day 6 post-weaning had abnormally shaped villi (tongue-shaped,
leaf-shaped, Kik et al, 1990).
Jejunal villus lengths (proximal and distal) and distal crypt depths at day 6 after
weaning were positively related to feed intake (Table 3b).
109
Chapter VI
Enzyme activities
Enzyme activities are presented in Table 4a (trypsin) and in Table 4b (chymotrypsin).
Four out of 20 piglets sampled at day 3 post-weaning had consumed less than 10
grams of feed daily and these piglets were analyzed separately because these piglets
('non-eaters') had higher enzyme activities in pancreatic tissue and higher
chymotrypsin activities per gram jejunal digesta than piglets that did consume
appreciable amounts of feed after weaning ('eaters') (Table 4a and 4b). The ratio
between trypsin and chymotrypsin activity in pancreas and jejunum was higher for
'eaters' than for 'non-eaters' (Table 4a).
At day 3, trypsin activity in the jejunum was affected by protein source (Table 4a).
The highest trypsin activity was found with piglets fed SMP and the lowest activity
was noticed in piglets fed SBM and FM.
A significant effect of feed intake within diet on chymotrypsin activities at day 3 postweaning was found (Table 4b). Chymotrypsin activity in pancreatic tissue was
positively related to feed intake only for piglets on the FM diet. Pancreatic
chymotrypsin activity and total chymotrypsin activity (pancreas + jejunum) were
higher for the FM fed piglets than for the soya fed piglets. Jejunal chymotrypsin
activity was higher for piglets fed SMP than for piglets fed FM.
At day 6, enzyme activities in pancreatic tissue and jejunal chyme were lowest for
piglets fed the FM diet compared to the other diets. Total enzyme activities
(pancreas + jejunum) were highest for the SPC fed piglets. Trypsin activity per gram
freeze-dried jejunal digesta was lower in piglets fed FM than in piglets fed SMP
(Table 4a). Chymotrypsin activity per gram freeze-dried jejunal chyme was lower in
piglets fed FM and SPC than in piglets fed SMP. Total enzyme activities in the
pancreas were highest for SPC fed piglets and lowest for FM fed piglets (Table 4a
and 4b). Total trypsin + chymotrypsin activities in pancreas + jejunum at day 6 were
positively related to post-weaning feed intake. This was mainly due to the strong
relation between feed intake and pancreatic enzyme activities (total and per gram
pancreatic tissue).
110
Chapter VI
Table 3a.
Effect of diet and feed intake
on organ weights (g and g/kg LW) and digesta
weight 5 (g) of newly weaned piglets (LSMeans (StdErr), b=coefficient of
regression)
day 0:
tissue weight (g)
tissue weight (g/kg)
digesta weight (g)
day 3:
organ weights:
stomach (g)
stomach (g/kg)
small intestine (g)
" (g/kg)
pancreas(g)
" (g/kgLW)
digesta weights:
stomach (g)
small intestine (g)
day 6:
organ weights:
stomach (g)
stomach (g/kg)
small intestine (g)
" (g/kg)
pancreas(g)
" (g/kg LW)
digesta weights:
stomach (g)
small intestine (g)
day 10:
organ weights:
stomach (g)
stomach (g/kg)
small intestine (g)
" (g/kg)
pancreas(g)
" (g/kgLW)
digesta weights:
stomach (g)
small intestine (g)
stomach:
38.2(3.1)
5.6 ( 0.2)
113.9(26.6)
small intestine:
345.5(21.4)
51.0( 1.9)
124.8 (28.9)
diet:
SMP
SPC
SBM
42.7
6.2
301
39.7
11.39
1.538b
41.8
5.9
264
38.4
11.46
1.66b
43.3
5.5
313
39.9
10.02
1.27a
43.2 0.9938
6.3 0.2393
280 0.5600
41.8 0.8598
10.73 0.6080
1.59b 0.0194
177.1
95
136.6
71
130.2
124
129.9 0.6722
79 0.2260
FM
p
54.5
54.4 48.7 47.2 0.3203
6.6
6.2
6.7
6.3 0.5402
370
405
362
355 0.4816
44.7
46.1
49.8
47.0 0.4835
13.47bc 15.41= 1.68ab 10.91a 0.0026
1.63
1.76
1.64
1.41 0.1954
125.0 204.7 202.1
158
258
214
62.9
6.7
481
51.6
15.62
1.67abc
198.7
208
pancreas
10.37 (0.84)
1.53(0.08)
feed intake:
b
P
0.023
-0.001
0.336
0.003
-0.006
-0.003
0.6522
0.6845
0.3001
0.9252
0.5970
0.0119
1.354 0.0017
0.797 0.0033
0.057
-0.003
0.657
0.009
0.038
0.002
0.0628
0.2918
0.0050
0.6534
0.0001
0.0189
191.8 0.2869
168 0.2398
-0.127 0.6490
-0.049 0.8774
60.3 0.8318
7.0 0.3218
421 0.6692
48.6 0.7757
13.17 0.1389
1.54a 0.0439
0.073 0.2154
-0.002 0.6047
0.924 0.0062
0.037 0.0873
0.019 0.1386
-0.000 0.8674
166.4 273.5 202.0 0.2966
185
188
130 0.6318
0.737 0.0721
-0.169 0.6529
57.3 64.7
6.5
7.4
438
459
49.9
51.7
17.59 14.52
1.98c 1.65ab
111
Chapter VI
Table 3b.
Effect of diet and feed intake
on digesta pH, ratio precipitable protein / crude
protein in the stomach, and gut wall morphology of newly weaned piglets
(LSMeans (StdErr), b=coefficient of regression)
day 0:
small intestine:
d jodenum
jejunum
ileum
5.82(0.1 8)
6.85(0.15)
7.47(0.12)
St omach:
3 80(0.46)
0.64 (0.04)
PH
pp/cp stomach
-
-
diet:
SMP
SPC
SBM
FM
P
day 3:
digesta pH:
stomach
6.38
duodenum
6.49
jejunum
7.10
ileum
7.37
pp/cp stomach 0.19
4.99
6.10
7.57
7.83
0.28
4.64
5.90
6.78
7.65
0.18
6.40
6.50
7.28
7.78
0.19
0.1821
0.1516
0.0743
0.6212
0.5874
0.012
-0.000
-0.007
-0.003
-0.000
0.1722
0.9484
0.0098
0.2388
0.2328
5.18
5.76
7.20
7.52
0.35
5.12
5.94
7.36
7.84
0.25
6.11 0.2550
6.22 0.8252
7.30 0.8835
7.87 0.3606
0.29 0.1825
-0.001
-0.002
-0.000
0.001
0.000
0.8299
0.5083
0.9626
0.4907
0.5441
301
280
286
300
296 0.7462
269 0.5551
0.400 0.0172
0.436 0.0131
139
130
129
144
137 0.5149
132 0.6271
0.043 0.3536
0.148 0.0431
3.89" 4.94a
6.08
5.91
7.70
7.75
7.96
7.89
0.57b 0.42ab
6.63b 0.0167
6.37 0.6338
7.02 0.1031
8.12 0.7991
0.27a 0.0266
day 6:
digesta pH:
stomach
4.29
duodenum
5.98
jejunum
7.20
ileum
7.71
pp/cp stomach 0.17
villus length (fim):
proximal
314
distal
300
crypt depth(urn):
proximal
132
distal
134
day 10:
digesta pH:
stomach
5.36ab
duodenum
5.94
jejunum
7.48
ileum
8.10
pp/cp stomach 0.40 ab
112
feed intake:
b
P
0.004
-0.001
-0.000
-0.000
0.000
0.4883
0.5715
0.8342
0.8005
0.3648
Chapter VI
Table 4a.
Effect of feed intake on trypsin activities in digesta and pancreas of newly
weaned piglets (b=coefficient of regression)
day 0:
Ptryp (trypsin activity per gram freeze-dried pancreatic tissue)
Jtryp (trypsin activity per gram freeze-dried jejunal digesta)
Ptryptot (total trypsin activity in pancreas)
Jtryptot (total trypsin activity in jejunum)
tottryp (total trypsin activity in pancreas + jejunum)
Pt/c (Ptryp/Pchym)
Jt/c (Jtryp/Jchym)
day 3:
Ptryp
Jtryp
Ptryptot
Jtryptot
tottryp
Pt/c
Jt/c
day 3:
'eaters'
Ptryp
Jtryp
Ptryptot
Jtryptot
tottryp
Pt/c
Jt/c
day 6:
Ptryp
Jtryp
Ptryptot
Jtryptot
tottryp
Pt/c
Jt/c
day 10:
Ptryp
Jtryp
Ptryptot
Jtryptot
tottryp
Pt/c
Jt/c
'non- •eaters'
3029
150
9239
370
9608
1.08
0.84
'eaters'
1225
268
2963
1547
4510
2.85
4.02
LSM StdErr
923
136
41
180
2193
483
272
979
3172
670
1.33
0.23
0.27
1.59
P
0.0002
0.2980
0.0001
0.0739
0.0025
0.0192
0.0009
diet:
SMP
SPC
SBM
FM
2025
602 c
5266
4149 b
9415 b
3.80
5.99
1068
360 b
2348
1524 a
3871 a
3.71
4.64
904
154 s
1974
1011 a
2985 a
2.29
3.13
1287
152 a
3325
954 a
4279 a
2.22
3.45
0.1572
0.0014
0.1923
0.0005
0.0153
0.1952
0.0666
12.64
0.57
31.78
5.76
37.54
0.01
0.01
0.0408
0.6089
0.0973
0.3710
0.0735
0.2601
0.3079
2526
2209 2041
790 b 649 a b 534 a b
7287 b 7803 b 5033 a b
4285
6269 4265
11571 b c 14072 c 9298 a b
5.94
4.44
4.52
5.52
5.18
7.61
1718
382 a
3832 a
3784
7615 a
6.36
7.98
0.5375
0.0389
0.0477
0.1346
0.0010
0.2082
0.0536
8.45
1.47
40.40
19.38
59.79
0.00
0.01
0.0212
0.0751
0.0003
0.0077
0.0001
0.4585
0.4571
4846
544
14435
3746
18180
6.15
6.42
0.0985
0.1261
0.3896
0.3285
0.7094
0.6862
0.2180
14.20
-0.99
60.10
2.48
62.58
0.01
-0.00
0.0237
0.2283
0.0086
0.8810
0.0114
0.4115
0.8065
3407
837
11327
8430
19757
6.77
7.00
2939
823
11665
7787
19452
5.44
8.29
2669
734
8912
7503
16415
6.70
6.48
feed intake:
b
113
Chapter VI
Table 4b.
Effect of feed intake on chymotrypsin activities in digesta and pancreas of
newly weaned piglets (b=coefficient of regression)
LSM StdErr
day 0:
Pchym (chymotrypsin activity per gram freeze-dried pancreatic tissue) 796
134
Jchym (chymotrypsin activity per gram freeze-dried jejunal digesta) 105
12
Pchymtot (total chymotrypsin activity n pancreas)
1899
421
Jchymtot (total chymotrypsin activity in jejunum)
560
85
totchym (total chymotrypsin activity in pancreas + jejunum)
2459
478
day 3:
Pchym
Jchym
Pchymtot
Jchymtot
totchym
SMP
feed intake
within diet
b
P
diet:
effect of:
day 3:
'eaters'
Pchym
Jchym
Pchymtot
Jchymtot
totchym
day 6:
Pchym
Jchym
Pchymtot
Jchymtot
totchym
1
p
0.0001
0.0008
0.0001
0.1520
0.0001
SPC
feed intake
within diet
b
p
-11.85
1.98
-55.26
2.49
-52.78
0.2422
0.2423
0.1150
0.7572
0.1571
-0.07
-0.48
-3.24
-3.97
-7.21
diet:
SMP
SPC
SBM
SBM
feed intake
within diet
b
p
0.9777 -8.20 0.0742
0.2496 0.26 0.7037
0.6802 -22.62 0.1243
0.0746 9.15 0.0263
0.4069 -13.48 0.3693
FM
feed intake
within diet
b
P
10.94
0.15
32.62
2.54
35.16
0.0008
0.6609
0.0015
0.1613
0.0015
p
feed intake:
b
P
432
532
442
269 0.1079
154b
95 a 104ab
51 a 0.0087
ab
b
a
1230
1913 1088
574a 0.0085
810
850
846
508 0.3374
2040bc 2763c 1934b 1082a 0.0028
1.52 0.0229
0.12 0.4580
7.84 0.0011
2.24 0.0990
10.08 0.0003
day 10:
Pchym
Jchym
Pchymtot
Jchymtot
totchym
2
*non-eaters'
2868
150
8992
199
9191
'eaters'
467
63
1138
369
1507
FM>SPC
FM>SPC, FM>SBM
536
124
1803
1195
2999
556
102
2190
868
3058
4
399
114
1285
1159
2443
FM
793 0.0756
84 0.3237
2368 0.1755
580 0.1283
2947 0.5082
FM>SPC, FM>SBM
114
1.75
-0.11
8.25
0.48
8.73
0.0644
0.4038
0.0212
0.7719
0.0127
diet
P
0.01481
0.2751
0.01812
0.03543
0.02164
Chapter VI
Discussion
Feed intake and growth
The low feed intake of piglets on the SMP diet may be related to the physical form
of the feed. Feeds containing large amounts of skimmilk powder are difficult to pellet
and will therefore result in hard pellets which are not readily accepted by young
piglets (Jensen, 1966, Liptrap & Hogberg, 1991). Pellets of the SMP diet were harder
than pellets of the SBM and FM diet (Table 1) and feed intake on the SMP diet was
lower during the first three post-weaning days. Feed intake on the SPC diet was
intermediate although pellets of this diet were very resistant to crumbling.
Piglets fed the SMP diet consumed less feed during the first three post-weaning days
than piglets fed the FM diet (Table 2) and at the same time had higher gastric
digesta weights (Table 3a). This may indicate that gastric emptying was faster in
piglets fed FM as was also found by Seve & Laplace (1975) with early weaned piglets
fed solid diets containing milk and fish proteins. This could have resulted in a more
regular feed intake for piglets fed the FM diet.
The differences in feed intake between diets did not result in differences in average
daily gain during the first three post-weaning days. Piglets fed the diets based on
animal proteins (SMP and FM) seemed to gain some liveweight during the first three
days post-weaning allthoug the differences between diets were not significant.
Average daily gain agreed with results of Bark et al. (1986), who studied feed intake
during the first week post-weaning in piglets weaned at 21 days of age.
Tissue weights
The relative weight of the empty stomach increased gradually after weaning, while
the relative weight of the small intestinal tissue decreased from weaning until three
days after weaning. The same trends were found by Kelly et al. (1991) with piglets
weaned at 14 days of age. The initial post-weaning decrease in relative small
intestinal weight may be related to the decline in feed intake in these animals
compared to intake during the suckling period. It could also indicate post-weaning
gut wall damage, because it was found that all piglets at day 6 had abnormally
shaped jejunal villi. The development of the gut wall (morphology, growth, secretion
and absorption) seems to be disturbed in the early post-weaning period.
The (relative) pancreatic weight of SPC and SMP fed piglets increased from weaning
until day 10. This is in accordance with the results of Kelly et al. (1991) with piglets
weaned at 14 days of age and fed a diet containing skim-milk powder, fish meal and
soyabean meal. Piglets on SBM and FM diets had the lowest relative pancreatic
weight at day 3 and day 6, respectively. These differences are difficult to explain,
115
Chapter VI
especially because they are not related to the content of antinutritional factors in the
diets.
Digesta pH
The increase in stomach pH after weaning is in accordance with data from Efird et
al. (1982) and Wilson & Leibholz (1981b). This result can be explained by postweaning feed intake pattern and/or the buffering capacity of the post-weaning diets.
A high pH in the stomach could lead to bacterial proliferation (Banwart, 1981) and
to disturbance of normal pepsin function (Kidder & Manners, 1978).
At day 10, piglets on the soya diets had lower pH in gastric contents than piglets on
the animal protein sources. This could be explained by the buffering capacity of the
diets, which was lower for the soya diets (Table 1).
Gastric pp/cp ratio
The changes in gastric pp/cp ratio after weaning indicate that the degree of gastric
protein breakdown is higher for solid diets than for sows milk. This could be
explained by the predominance of chymosin compared to pepsin activity in suckling
piglets. Before weaning, coagulation of milk proteins (clot formation) is more
important than protein hydrolysis (Cranwell & Moughan, 1989). Between day 3 and
10 an increase in gastric pp/cp ratio was found, indicating changes in gastric protein
breakdown and/or changes in gastric emptying patterns.
At day 10, gastric pp/cp ratio was lower for the FM fed piglets than for the SPC fed
piglets, indicating a higher degree of gastric protein breakdown with the FM diet.
It is clear that the extent of gastric protein hydrolysis depends on age (or time after
weaning) and dietary composition (protein source). This was also found by Leibholz
(1986) who reported that piglets aged 28 days had higher stomach pH and less
gastric protein breakdown than older piglets.
Gut wall morphology
The gut wall damage as reflected by the abnormally shaped villi at 6 days postweaning is a common finding in literature (Hampson, 1986, Deprez et al, 1987, Cera
et al, 1988).
Villus lengths were comparable to results from Deprez et al. (1987) of piglets at day
6 after weaning onto a dry diet and from Hampson (1986) with piglets at day 5 after
weaning and from Miller et al. (1986) with piglets at one week after weaning.
Crypt depths were slightly smaller than data reported by Hampson (1986), Miller et
al. (1986), Deprez et al. (1987) and by Kelly et al.(1991).
The positive relation of villus lengths and (distal) crypt depths with feed intake as
116
Chapter VI
found in this experiment is not known from literature. This relation could indicate a
stimulatory effect of feed intake on development of the gut wall.
It was proposed by Gall & Chung (1982) in rabbits and by Cera et al. (1988) in pigs
that low feed intake during the early post-weaning period may be a contributing
factor to reduced villus height. However, Kelly et al. (1991) also found reductions in
villus height in piglets fed through gastric intubation to maintain continuous nutrient
supply.
Enzyme activities
The development of trypsin and chymotrypsin activities in pancreas and jejunum after
weaning strongly depended on dietary protein source and post-weaning feed intake.
At day 3, 'non-eaters' apparently stored large amounts of trypsin and chymotrypsin in
their pancreatic tissue without substantial secretion into the gut. Intestinal substrate
availability seems to be involved in the stimulation of pancreatic trypsin and
chymotrypsin secretion as was suggested by DiMagno et al. (1973), Niederau et al.
(1986) and Valette et al. (1992).
This mechanism is stimulated through the digestive endproducts of intestinal protein
digestion (Grendell & Rothman, 1981, Valette et al, 1992). DiMagno et al. (1973)
found that essential amino acids infused into the duodenum or jejunum of humans
stimulated pancreatic enzyme secretion. They postulated, that the products of protein
digestion after absorption inhibit pancreatic enzyme secretion through glucagon
release. The study by Niederau et al. (1986) showed that arginine and lysine (the sites
of tryptic cleavage) specifically caused the release of trypsinogen in pancreatic tissue
homogenate whereas phenylalanine and tryptophan (sites of chymotryptic cleavage)
caused release of trypsinogen and chymotrypsinogen.
Valette et al. (1992) found in experiments with pancreas-cannulated growing pigs fed
diets based on either casein or rapeseed that dietary protein source influences
pancreatic enzyme secretion.
Skimmilk powder appeared to be the strongest stimulant of trypsin synthesis and
secretion during the first three days post-weaning. At day 6, high pancreatic and
jejunal trypsin activities were found with the SPC fed piglets, while at day 10, the
effect of dietary protein source on trypsin activities had disappeared. Feed intake was
positively related to pancreatic trypsin activity and therefore might affect trypsin
synthesis.
The interaction between dietary protein source and feed intake during the first three
post-weaning days with respect to (especially pancreatic) chymotrypsin activities is
striking. Only piglets on FM (= piglets consuming on average more than 100 grams
feed per day during the first three post-weaning days) showed a positive relation
117
Chapter VI
between feed intake and pancreatic chymotrypsin activity.
It is evident, that piglets on the FM diet had the highest feed intake during the first
three days post-weaning. This could imply a more regular development of feed intake
of newly weaned piglets on the FM diet. It could also imply a better development of
the enzyme system compared to piglets on the other three diets. At day 6, lowest
chymotrypsin activities were found with the FM diet and highest with the SMP and
SPC diets. By day 10 the differences between diets had disappeared, while feed
intake was still related to total pancreatic chymotrypsin activity.
The trypsin/chymotrypsin ratio in pancreas and jejunum was lower for 'non-eaters'
than for 'eaters'. This is in accordance with the findings from Corring et al. (1978),
Efird et al. (1982), Owsley et al. (1986) and Lindemann et al. (1986) who state that
chymotrypsin is the predominant pancreatic protease during the suckling period,
while trypsin increases specifically after weaning. Our results indicate that the shift
from chymotrypsin to trypsin is related to post-weaning solid feed intake rather than
to weaning itself.
Conclusions
From the results of this experiment it can be concluded that post-weaning feed intake
is affected by type of post-weaning diet. Dietary protein source also influenced
pancreatic tissue weight and trypsin and chymotrypsin activities in pancreatic tissue
and jejunal chyme.
In piglets fed SPC or FM, low pancreatic tissue weight coincided with low jejunal
enzyme activities.
Piglets fed the SMP diet had low feed intakes but higher gastric digesta weights
compared to piglets fed the FM diet. This suggests a higher rate of gastric emptying
when FM was fed. A faster gastric emptying combined with a higher gastric pH leads
to a more regular supply of more alkaline digesta to the duodenum. From this it can
be expected that the pancreas is less challenged to secrete bicarbonate into the gut
lumen. This hypothesis is supported by the finding that pancreatic tissue weight was
also lower for piglets fed the FM diet. The low enzyme activities in the jejunum of
piglets fed FM could reflect a lower need for pancreatic enzymes when gastric
emptying occurs more gradually. Dietary buffering capacity, gastric protein hydrolysis
and gastric emptying seem to be important factors in the digestion of different
protein sources by newly weaned piglets.
Higher feed intake was positively related to villus length at day 6 after weaning. This
finding merits further study as villus atrophy is an important consequence of weaning
and might account for considerable endogenous nitrogen losses in newly weaned
piglets.
118
Chapter VI
From the results presented herein it can de derived that feed intake as well as
dietary composition are important factors for the development of the digestive organs
of newly weaned piglets. Dietary fish meal had a stimulatory effect on the
development of post-weaning feed intake, probably related to modifications of gastric
emptying patterns related to intestinal protein digestion through pancreatic
secretions.
Acknowledgements
The authors wish to thank Mr H. Schipper of the Department of Experimental
Animal Morphology and Cell Biology for the assistance with the measurement of
villus heights and crypt depths.
References
Baak, M.J. van, Rietveld, E.C., Makkink, C.A., 1991.
Determination of trypsin and chymotrypsin activity in pancreatic juice: the
effect of freeze-drying and storage. In: Proc. Vth Intern. Symp. on Dig.Physiol
in Pigs, pp. 356-360 [M.WA. Verstegen,J. Huisman and L~A. den Hartog,
editors]Wageningen(Doorwerth), Netherlands.EAAP Publicationno 54,1991.
Banwart, G.J., 1981.
Basic Food Microbiology.
TheAVI PublishingCompany, Westport, Connecticut, USA.
Bark, L.J., Crenshaw, T.D., Leibbrandt, V.D., 1986.
The effect of meal intervals and weaning on feed intake of early weaned pigs.
/. Anim. Sci. 62:1233-1239
Bergmeyer, H.U., 1974.
Methoden der Enzymatischen Analyse. Third Edition. Weinheim: Verlag
Chemie.
Cera, K.R., Mahan, D.C., Cross, R.F., Reinhart, G.A., Whitmoyer, R.E., 1988.
Effect of age, weaning and postweaning diet on small intestinal growth and
jejunal morphology in young swine./. Anim. Sci. 66:574-584
Corring, T., Aumaitre, A., Durand, G., 1978.
Development of digestive enzymes in the piglet from birth to 8 weeks. Nutr.
Metab.22:231-243
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Chapter VI
Cranwell, P.D., Moughan, P.J., 1989.
Biological limitations imposed by the digestive system to the growth
performance of weaned pigs.In: ManipulatingPigProductionII. Proceedings of
the Biennial Conference of the Australasian Pig ScienceAssociation (A.P.S-A.),
pp. 140-159[J.L. Bamett and D.P.Hennessy,editors]. Albury, New South Wales,
Australia.
Deprez, P., Deroose, P., Van der Hende, C , Muylle, E., Oyaert, W., 1987.
Liquid versus dry feeding in weaned piglets: the influence on small intestinal
morphology./. Vet.Med. B34-.254-259
DiMagno, E.P., Go, V.L.W., Summerskill, W.H.J., 1973.
Intraluminal and postabsorptive effects of amino acids on pancreatic enzyme
secretion./. Lab. Clin.Med.82(2):241-248
Efird, R.C., Armstrong, W.D., Herman, D.L., 1982.
The development of digestive capacity in young pigs: effects of age and
weaning system./. Anim. Sci. 55(6):1380-1387
Gall, D.G., Chung, M , 1982.
Effect of body weight on postnatal development of the proximal small
intestine of the rabbit. Biol. Neonate42:159-165
Grendell, J.H., Rothman, S.S., 1981.
Digestive end products mobilize secretory proteins from subcellular stores in
the pancreas.Am. J.Physiol. 241:G67-G73
Hampson, D.J., 1986.
Alterations in piglet small intestinal structure at weaning. Res. Vet. Sci.
40(l):32-40
Jensen, A.H., 1966.
Pelleting rations for swine.Feedstuffs 38(31):24-27
Kidder, D.E., Manners, M.J., 1978.
Digestion in the Pig. Scientechnica,Bristol, UK.
Kelly, D., Smyth, J.A., McCracken, K.J., 1991.
Digestive development of the early-weaned pig. 1. Effect of continuous
nutrient supply on the development of the digestive tract and on changes in
digestive enzyme activity during the first week post-weaning. Br. J. Nutr.
65:169-180
Kik, M.J.L., Huisman, J., Van der Poel, A.F.B., Mouwen, J.M.V.M., 1990.
Pathological changes of the small intestinal mucosa of piglets after feeding of
Phaseolus vulgaris beans. Vet.Pathol. 27:329-334
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Leibholz, J., 1986.
Some aspects of digestion in the pig from birth to 56 days of age. Proc.Nutr.
Soc. (Aust.)11:32-39
Lindemann, M.D., Cornelius, S.G., El Kandelgy, S.M., Moser, R.L., Pettigrew, J.E.,
1986.
Effect of age, weaning and diet on digestive enzyme levels in the piglet. /.
Anim. Sci. 62:1298-1307
Liptrap, D.O., Hogberg, M.G., 1991.
Physical forms of feed: feed processing and feeder design and operation. In:
Swine Nutrition, pp. 373-386 [E.R. Miller,D.E. Ullrey and AJ. Lewis,editors].
Butterworth-Heinemann, Boston, USA.
Miller, B.G., James, P.S.Smith, M.W., Bourne, F.J., 1986.
Effect of weaning on the capacity of pig intestinal villi to digest and absorb
nutrients.J. Agric.Set, Cambridge 107:579-589
Niederau, C, Grendell, J.H., Rothman, S.S., 1986.
Digestive end products release pancreatic enzymes from particulate cellular
pools, particularly zymogen granules.Biochim. Biophys. Acta 881:281-291
Okai, D.B., Aherne, F.X., Hardin, R.T., 1976.
Effects of creep and starter composition on feed intake and performance of
young pigs. Can.J.Anim. Sci. 56:573-586
Owsley, W.F., Orr, D.E., Tribble, L.F., 1986.
Effects of age and diet on the development of the pancreas and the synthesis
and secretion of pancreatic enzymes in the young pig./. Anim. Sci. 63:497-504
SAS Institute Inc., 1990.
SAS Procedures Guide. Version6, ThirdEdition. Cary,NC: SAS Institute Inc.,
705pp.
Seve, B., Laplace, J.P., 1975.
Influence de la substitution des proteines du lait par des proteines de poisson
sur quelques caracteristiques du contenu gastrique chez le porcelet sevre a 12
jours (Influence of the substitution of milk proteins by fish proteins on some
characteristics of the gastric content in the piglet weaned at 12 days). Ann.
Zootechnie 24(l):43-57
Souffrant, W.B.,1991.
Endogenous nitrogen losses during digestion in pigs.In: Proceedings of the Vth
International Symposium on DigestivePhysiology in Pigs.pp. 147-166 [M.WA.
Verstegen, J. Huisman and LA. den Hartog,editors] Wageningen(Doorwerth),
Netherlands.EAAP PublicationNo 54,1991.
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Ternouth, J.H., Roy, J.H.B., Siddons, R.C., 1974.
Concurrent studies of the flow of digesta in the duodenum and of exocrine
pancreatic secretion of calves. 2. The effects of addition of fat to skim-milk
and of 'severe' preheating treatment of spray-dried skim-milk powder. Br. J.
Nutr. 31:13-26
Valette, P., Malouin, H. Corring, T., Savoie, L., Gueugneau, A.M., Berot, S., 1992.
Effects of diets containing casein and rapeseed on enzyme secretion from the
exocrine pancreas in the pig.Br.J.Nutr. 67:215-222
Wilson, R.H., Leibholz, J., 1981a.
Digestion in the pig between 7 and 35 days of age. 1.The performance of pigs
given milk and soyabean proteins.Br.J.Nutr. 45:301-319
Wilson, R.H., Leibholz, J., 1981b.
Digestion in the pig between 7 and 35 days of age. 2. The digestion of dry
matter and the pH of digesta in pigs given milk and soyabean proteins. Br.J.
Nutr. 45:321-336
Wilson, R.H., Leibholz, J., 1981c.
Digestion in the pig between 7 and 35 days of age. 3. The digestion of
nitrogen in pigs given milk and soyabean protein. Br.J.Nutr. 45:337-346
122
ChapterVII
A NOTE ON THE RESPONSE OF PANCREATIC JUICE SECRETION
TO DIFFERENT STIMULANTS
IN YOUNG PIGLETS
Caroline A. Makkink1, J. Martje Fentener van Vlissingen2,3, Marlou W. Bosch1,
Tamme Zandstra1, Martin W.A. Verstegen1
1
2
Agricultural University, Department of Animal Nutrition,
Haagsteeg 4,6708 PM Wageningen, TheNetherlands
TNO-Institute for Animal Nutrition andPhysiology (ILOB),
P.O. Box15,6700 AA Wageningen, TheNetherlands
3
Present address: TNO-Institute for Experimental Biology,
P.O. Box360, 3700 AJ Zeist, TheNetherlands
123
Chapter VII
Introduction
Pancreatic enzyme activities are often reported to decline after weaning (Lindemann
et al, 1986, Owsley et al, 1986). It is suggested (Jones, 1986) that this decline may
induces disturbed (protein) digestion in newly weaned piglets. This might account for
the post-weaning lag often observed in pigs. The usually low feed intake after
weaning could also be responsible for the low digestive enzyme activities.
The regulation of pancreatic enzyme secretion, especially during the adaptation phase
just after weaning, is insufficiently clear yet. Pancreatic secretion is known to be
stimulated by the presence of (hydrolyzed) dietary components in the small intestine.
This stimulation is mediated through hormones and peptides produced by the
intestinal wall (Solomon, 1987).
When pancreatic enzyme secretion is low, this could be due either to low
responsiveness of the pancreas towards stimulatory hormones (secretin, CCK) or to
diminished release of these hormones.
The purpose of this study was to investigate the response of pancreatic secretion in
young piglets to exogenous stimulation by either intravenous hormone injection or an
intraduodenal infusion of a hydrolyzed casein solution of pH 2.0.
A method was developed and two trials were performed to study these responses in
anaesthetized piglets before and after weaning.
Materialsand Methods
trial 1
Five piglets from one litter were weaned at AVi weeks of age (no access to creep feed
during the suckling period), housed individually and fed one of two experimental
diets (Table 1) based on either skim milk powder (SMP) or soyabean meal (SBM).
The piglets received the diets at a daily amount of 4% of their live weight, divided in
two portions. At 8.00 am, 200 gram was fed and the rest at 4.30 pm. At 8V2 weeks of
age, the piglets were anaesthetized (0 2 , N 2 0, halothane) one hour after the morning
feeding without premedication. The external jugular vein and the pancreatic duct
were catheterized (catheter diameter 2 mm). After surgery, the piglets were kept
under anaesthesia while collections of pancreatic juice were made. After two hours of
Tjasal secretion collection', an intravenous injection of secretin (Sekretolin, Hoechst,
Frankfurt am Main, Germany) was given (0.1 clinical unit(~ 8 pmol) per kg live
weight). The dose injected was within the range of doses used by Harada et al.
124
Chapter VII
(1988). After secretin injection, stimulated pancreatic juice secretion was collected for
another half hour. Hereafter, the piglets were euthanized (T61,intravenously) and
the pancreas was removed.
Table 1.
Composition of experimental diets
trial 1
trial 2
SMP
ineredient
soybean meal (47%CP)
skimmed milk powder (35%CP)
maize starch
dextrose
lactose
sunflower/soy oil
cellulose
vit/min premix
calculated contents
crude protein
crude fat
crude fibre
inorganic matter
net energy (kcal/kg)
SBM
SMP
-
34.40
-
45.50
29.60
15.00
-
47.00
29.50
13.44
39.84
15.00
SBM
34.40
-
-
-
-
2.00
5.00
1.00
2.00
2.85
1.00
2.00
5.00
1.00
20.88
10.96
22.50
2.75
2.85
1.00
16.08
2.49
4.95
5.33
2555
16.37
2.44
5.02
5.88
2477
16.21
2.51
4.95
5.46
2440
16.38
3.18
5.02
5.99
2443
trial 2
Eight piglets from one litter with no access to creep feed during the suckling period
were used in this trial. At 23 days of age, two suckling piglets were anaesthetized and
catheterized as described for trial 1.An additional catheter was placed in the
duodenum. Pancreatic juice was collected for two hours. Hereafter, 25 ml of a
hydrolysed casein (casein enzymatic hydrolysate, Sigma Chemical Co, St Louis, USA)
solution (5 gram casein per 100 ml, pH 2.0) was infused into the duodenum and
another two hours of pancreatic juice collection followed. Afterwards, the pancreas
was removed and the piglets euthanized (T61,intravenously).
The remaining six piglets were weaned at 26 days of age, individually housed and fed
one of two diets based on either skimmed milk powder (SMP) or soybean meal
(SBM). At 32 days of age, the piglets (three on SMP and three on SBM) were
anaesthetized, catheterized, sampled and euthanized as described above.
125
Chapter VII
Trypsin and chymotrypsin activities were assessed in pancreatic juice and freeze dried
pancreatic tissue according to Bergmeyer (1974).Trypsinogen and chymotrypsinogen
were activated by enterokinase (Sigma Chemical Company, St. Louis, USA) before
analysis.
Results
No health problems were encountered between weaning and sampling of the piglets.
trial 1
In trial 1, all catheterizations and collections of pancreatic juice were successful. Live
weights, pancreatic weight and pancreatic juice volume secretion are presented in
Table 2. No effect of diet could be detected for any of the parameters measured.
The basal pancreatic juice secretion was 0.36 ml per hour and was not affected by
diet. Pancreatic juice secretion increased more than 20-fold after stimulation with
intravenous secretin.
Table 2.
Live weights, pancreatic weightsand juice secretion.Trial 1.
age (days)
diet
number of piglets
live weight (kg)
pancreas weight (g)
pancreas weight (g/kg LW)
basal secretion (ml/h)
stimulated secretion (ml/h)
56-60
SMP
2
SBM
3
15.0
27.8
1.85
0.36
8.40
13.3
23.5
1.76
0.36
10.0
VMSE
1.25
4.71
0.33
0.19
2.87
trial 2
In trial 2, collections could not be made in one piglet (at day 23) due to surgical
problems. Live weights, pancreatic weight and pancreatic juice secretion (volume and
enzyme activities) are presented in Table 3.
No significant effects of diet or age could be demonstrated.
Intraduodenal infusion of an acid solution of hydrolyzed casein resulted in a trhreeto five-fold increase in pancreatic juice secretion (Table 3). Enzyme activities in
126
Chapter VII
pancreatic juice (units per ml) decreased upon stimulation with hydrolyzed casein of
pH 2.0 (Table 3). Enzyme secretion (units per hour) was not significantly changed
after stimulation.
Table 3.
Live weights, pancreatic weights,juice secretion and enzymeactivities.Trial
2.Enzymeactivities inpancreatic tissueare expressed asunits pergram
freeze-dried tissue.
age (days)
diet
number of piglets
23
32
SMP
3
-
2
live weight (kg)
pancreas weight (g)
pancreas weight (g/kg LW)
7.1
5.9
0.82
basal secretion (ml/h)
trypsin act. (U/ml)
trypsin act. (U/h)
chymotrypsin act. (U/ml)
chymotrypsin act. (U/h)
stimulated secretion (ml/h)
trypsin act. (U/ml)
trypsin act. (U/h)
chymotrypsin act. (U/ml)
chymotrypsin act. (U/h)
trypsin act. (U/g pancreas)
chymotrypsin act. (U/g pancreas)
SBM
3
VMSE
8.7
9.2
1.07
1.66
2.57
0.36
0.25
401
100
62
15
0.24
0.23
1237 1801
280
553
538
345
128
87
0.17
789
484
292
102
0.85
204
173
52
44
0.77
1.21
389
793
447
503
107
104
118
57
0.61
282
317
72
61
1918
1134
8.5
12.2
1.47
3200
968
3998
815
998
385
Discussion
The basal pancreatic juice secretion in these trials is approximately ten-fold lower
than volumes found by Pierzynowski et al. (1988).After secretin stimulation
pancreatic juice secretion was also lower than reported by Pierzynowski et al. (1988).
These differences may be due to the fact that the dose of secretin used in our study
was five times lower than the dose used by Pierzynowski et al. (1988). Another
possible explanation lies in the fact that our piglets were anaesthetized which may
have decreased the responsiveness of the pancreas.
Despite the relatively low dose of secretin, all piglets clearly responded to the
127
Chapter VII
injection with an increase in pancreatic juice secretion. From trial 1it can be
concluded that 8 weeks old piglets are capable of increasing the pancreatic secretion
in response to intravenous secretin injection. From trial 2 it appears that even before
weaning the pancreas responds to an intraduodenal load of hydrolysed casein of low
pH. Upon duodenal infusion of the test-solution (trial 2), a more diluted pancreatic
juice is secreted after weaning: the volume is increased three- to five-fold, while
trypsin secretion (Units/ml) is decreased two- to three-fold and chymotrypsin
secretion (Units/ml) is decreased three- to five-fold. Enzyme secretion in units per
hour hardly changes after duodenal administration of a hydrolyzed casein solution of
pH 2.0.
These trials show that young piglets are capable of changing their pancreatic
secretion in response to stimulants. It appears that a decline in pancreatic secretion
as found by Lindemann et al. (1986) and by Owsley et al. (1986) is not due to an
inability of the pancreas to respond to stimulants.
Anaesthetized piglets fitted with a pancreatic duct catheter can provide a useful
model to study the response of pancreatic secretion to various stimulants
(intravenously, intragastric or intraduodenally).
References
Bergmeyer, H.U., 1974.
Methoden der Enzymatischen Analyse. ThirdEdition. Weinheim: Verlag
Chemie.
Harada, E., Kiriyama, H., Kobayashi, E., Tsuchita, H., 1988.
Postnatal development of biliary and pancreatic exocrine secretion in piglets.
Comp. Biochem. Physiol. 91A(1):43-51
Jones, E.E., 1986.
Biological development and nutritional requirement of the neonatal pig.
Proceedings of the Georgia Nutrition Conference1986. pp 145-162
Lindemann, M.D., Cornelius, S.G., El Kandelgy, S.M., Moser, R.L., Pettigrew, J.E.,
1986.
Effect of age,weaning and diet on digestive enzyme levels in the piglet. /.
Anim. Sci. 62:1298-1307
Owsley, W.F., Orr, D.E., Tribble, L.F., 1986.
Effects of age and diet on the development of the pancreas and the synthesis
and secretion of pancreatic enzymes in the young pig.J. Anim. Sci. 63:497-504
128
Chapter VII
Pierzynowski, S.G., Westrom, B.R., Karlsson, B.W., Svendsen, J., Nilsson, B.,
1988.
Pancreatic cannulation of young pigs for long-term study of exocrine
pancreatic function. Can.J.Anim. Set 68:953-959
Solomon, T.E., 1987.
Control of Exocrine Pancreatic Secretion. Chapter43 in:Physiology of the
GastrointestinalTract. Second Edition. Volume 2 (Eds. L.R. Johnson,J.
Christensen, M.J.Jackson, E.D.Jacobson,J.H. Walsh).Raven Press, New York,
USA.pp 1173-1207
129
Chapter VIII
GENERAL DISCUSSION
131
Chapter VIII
Introduction
The main objective of the present investigationswasto study the development of protein
digestive capacity inyoungweaned piglets.Protein isthe most expensive feed ingredient
and especially young piglets have difficulties digesting non-milk proteins (Wilson and
Leibholz, 1981).As pointed out inChapter I,the digestion of non-milk proteins requires
a physiological adaptation of the digestive functions of the newly weaned piglet. This
adaptation may be more difficult when the dietary protein source is less similar to sow
milk protein.
To investigate the development ofpancreatic secretion after weaning itwould have been
useful to fit piglets with a pancreatic cannula. This kind of surgery had been applied in
older swine, however, it did not prove succesful in young piglets. Therefore, this
technique was abandoned and slaughter experiments were designed.
For practical and statistical reasons, the experiments described in this thesis have been
performed with individually housed piglets. It was shown with fattening pigs (De Haer,
1992)that housing system affects the feed intake patterns and growth of the animals. In
individualhousing systemslesscompetition willoccur,buton theother hand thepigsmay
be less encouraged to start eating by observing their 'room-mates' eat (De Haer, 1992).
The impact of these influencing factors on the performance of young piglets during the
early post-weaning period is unknown. Individual housing probably aggravates postweaning social stress in young piglets, thereby increasing activity and lowering piglet
performance as compared to group housing.
In Chapter I protein digestion in piglets is described and it is concluded that differences
in protein digestibility occur between protein sources. These differences are related to
the age of the piglets.Youngpigletshavegreater difficulties digestingplant proteins than
older piglets. In other words, the differences in digestibility diminish with advancing age
of the piglets (e.g., Wilson and Leibholz, 1981).
From this finding, several research questions and issues were formulated (Chapter I).
Different aspects of protein digestive capacity of newlyweaned pigletswere investigated
and described inthis thesis.Inthischapter these aspectswillbe considered and discussed
inan integrated approach combiningtheresultsobtained from the experiments described
in the previous chapters.
132
Chapter VIII
1.
Isthedifferenceinapparent(ilealandfaecal) nitrogendigestibilitybetween vegetable
protein and milk protein in young piglets caused by the differentprotein sources
(dietaryfactors) orrelatedtoendogenousnitrogen losses(interaction betweendietary
and animal factors)?
Digestibility of protein isusually measured as 'apparent faecal' N (nitrogen) digestibility,
calculated as follows:
Ndig = Nfeed - Nfaece8
Nfeed
Nfeed = N in feed (g/d)
N
fae«8 = N in faeces (g/d)
Thus, also 'apparent ileal' N digestibility can be measured. This is done by using
cannulated pigsand replacing NfaecM byNilealchyme-Faecesor ilealchymecanbe collected
quantitatively (24 hours a day) to obtain the amount of N excreted per day. Another
possibility is to include an indigestible marker in the feed (Cr 2 O s , Co-EDTA or others)
which can be used to calculate daily N excretion when faeces or chyme is not collected
quantitatively.
Zebrowska (1973), Just et al. (1981) and Darcy et al. (1983) showed that proteins
digested by the microflora in the caecum or colon are of no benefit to the pig because
they do not contribute to body protein synthesis. Nitrogen in the large intestine may be
primarily absorbed as urea or utilized by intestinal bacteria for microbial protein
synthesis.Dietaryfactors mayaffect the degree ofurea formation and absorption and the
synthesis of microbial protein.
Because of the confounding action of bacteria in the large intestine with regard to
apparent nitrogen digestibility, it is recommended to determine nitrogen digestibility in
pigs at the terminal ileum.
Nitrogen collected at theterminal ileum consistsnot onlyof exogenous (dietary) nitrogen
but also of endogenous nitrogen originating from the pig such as digestive enzymes,
mucus and sloughed off gut wall cells. Also bacteria are present at the terminal ileum
that will utilize nitrogen from both endogenous and exogenous sources. These
confounding aspects result in 'apparent' N digestibility as opposed to 'true' or 'real' N
digestibilitywhen corrections are made for the amount of endogenous N present in ileal
chyme or faeces.
To unravel the causes of poor digestibility of plant proteinsbyyoung piglets a distinction
should be made between apparent and true ileal N digestibility.
The lsN-isotope dilution technique was used to determine endogenous N losses at the
terminal ileum of piglets fed diets based on different protein sources (Chapter II). The
133
Chapter VIII
protein sources used in this study, skim milk powder, soyabean meal, isolated soya
protein and fish meal,were chosen for their practical application in piglet feeds and for
their range in apparent nitrogen digestibility (Chapter I).
The prerequisites of the 16N-isotope dilution technique have been discussed by Schulze
et al. (1993).An important aspect of the l5N-isotope dilution technique is the choice of
an appropriate substance to serve as a reference pool for endogenous N secretion. This
is necessary because it is extremely difficult and in some cases until now impossible to
obtain direct samples of each precursor pool separately. From the study of Schulzeet al
(1993) it appears that the TCA-soluble fraction of the blood plasma seems to be the
most suitable reference pool for the determination of endogenous N losses.
From the results presented in Chapter II it was concluded that the differences in
endogenous N losses between piglets fed the different protein sources were the main
cause of the differences in apparent N digestibilities. Small differences in true ileal
protein digestibility were found between isolated soya protein and the other protein
sources, but all four protein sources were highly digestible (true ileal digestibility
coefficients > 0.9).Similar resultswere obtained byWalker etal.(1986) using four week
old piglets fed diets based on casein or various soya products and determining
endogenous N losses by feeding a hydrolyzed casein diet.
Skim milk powder resulted in low endogenous N losses (less than 800 mg per day),
similar to endogenous Nlosseswhen nitrogen free diets are fed toyoung piglets (Wilson
and Leibholz, 1981).
The protein sources used in this study which are also applied in practical piglet feeding
all had high true ileal nitrogen digestibilities. It may be expected that other protein
sources have lower true nitrogen digestibilities as was shown by Huisman (1990) with
youngpigletsfed commonbeans (Phaseolusvulgaris) asthesolesourceofdietary protein.
The experiment described inChapter IIwasperformed with pigletsof ± 7weeks of age,
weaned at 3'/2weeks of age.In other words,these pigletswere probably fully adapted to
the experimental diets. It can be expected that the differences between diets are more
pronounced when adaptation is not fully established as in the case of newly weaned
piglets. More research is needed to study the development of apparent and true
digestibility ofvarious feed components duringadaptational phases, e.g., during the early
post-weaning period. This may, however, prove difficult because of the surgery and
subsequent recovery periods needed to perform this type of experiment.
In the future we need to identify the contribution of the different sources of endogenous
nitrogen recovered from the ileum when non-milk proteins are fed. This may improve
and extend our knowledge on protein digestion in young piglets with regard to the site
where interactions between dietary components and the gastro-intestinal tract occur. It
may also provide tools to improve the digestion of non-milk proteins by newly weaned
134
Chapter VIII
pigs when the specific reactions of the animal in response to specific dietary factors is
known in more detail.
From Chapter IIit isclear that dietary protein source affects endogenous nitrogen losses
at the terminal ileum ofyoungpiglets.Differences inapparent nitrogen digestibilitywere
mainly caused by differences in endogenous nitrogen losses when diets based on skim
milk powder, isolated soya protein, soyabean meal or fish meal were fed. The specific
sources of endogenous nitrogen could not be distinguished in this experiment. The
pancreas might be an important secretory gland in this respect because of its key role in
protein digestion.
2.
Thepancreas isimportantfor thesecretion ofproteolyticenzymes.Some aspects of
pancreaticsecretion inpigsand the effectsof dietaryfactors onprotein and enzyme
secretion (obtainedfromliterature)
The pancreas is an important secretory gland for the digestion of protein. Many
investigations have been reported in literature on the impact of pancreatic enzyme
secretion on (protein) digestion inpigs (e.g.Pekasetal, 1964;Corring, 1977;Zebrowska
et al, 1983;Hee, 1984).
Several authors studying the enzyme development of newly weaned pigs have found a
decrease inpancreatic enzyme activities after weaning (Hartman etal, 1961; Lindemann
etal, 1986;Owsleyetal, 1986).From these findings it has been suggested (Jones, 1986)
that (pancreatic) digestive enzyme capacity might be alimiting factor inprotein digestion
bynewlyweaned piglets.A lack of pancreatic proteolytic enzymes mayhamper intestinal
protein digestion and maythus lead toincreased amounts of nutrients entering the ileum
and large intestine. This may enhance fermentation, proliferation of pathogens and
probably digestive disorders (diarrhoea). This hypothesis deserves further study because
a shortage of pancreatic enzymes could be compensated for by adding enzymes to the
piglet diet.
Because of the importance of the pancreas both as a source of endogenous nitrogen and
as a digestive gland producing proteolytic enzymes, it was decided to study the effect of
diet on the development of pancreatic secretion in newly weaned piglets.
In Chapter III a review is made of literature on pancreatic secretion in pigs. It is clear
from literature that pancreatic enzyme secretion depends on the composition of the diet
(amount and type of protein, fat and carbohydrate), on age of the pig and on stress
factors (weaning).
As stressed in Chapter III, the pancreatic enzyme activities in small intestinal contents
asmeasured inthelaboratory areusuallymanyfold higherthanthetheoretically required
135
Chapter VIII
activity for digestion of standard pig feeds (Corring, 1982;Zebrowska etal, 1983). The
fact that digestive enzyme levels adapt to dietary changes illustrates that the
overproduction isonlyapparent, not true.This apparent overproduction isprobably due
to the suboptimal conditions inthe gutlumen withregard to temperature, pH, substrate
availability and presence of interfering substances (e.g. protease inhibitors) which may
decrease the actual 'in vivo' enzyme activity compared to the enzyme activity measured
'in vitro' (under optimal conditions). In Figure 1the effect of buffer pH on trypsin and
chymotrypsin activities is illustrated. A one unit deviation from the optimum pH value
leads to a considerable reduction in enzyme activity.
The causes of the apparent overproduction of digestive enzymes should be studied in
more detail. It isclear that part of the apparent overproduction isneeded to account for
suboptimal digesta pH. The different types of substrates presented to the digestive
enzymes 'in vivo' probably also require more enzyme activity than needed for the
hydrolysis of specific synthetic substrates employed in 'in vitro' enzyme analyses. The
syntheticsubstrates (TAME for trypsin analysisand BTEE for chymotrypsin analysis) are
selected for their affinity for the respective enzymes.
chymotrypsin
trypsin
300
L,
200
100 -
pHof buffer
Figure 1.
Trypsin (Merck, 40 U/mg, porcine) and a-chymotrypsin (Merck, 350 U/mg,
bovine) activity (AA) asaffected by pH of the buffer solution (Makkink, 1993,
unpublished results).Standard trypsin and chymotrypsin analyses are performed
at pH 8.1 and 7.8,respectively.
136
Chapter VIII
Not much is known about the kinetics of hydrolysis of pancreatic enzymes during the
passage through the digestive tract. The timing of enzymatic digestion can also play an
important role when considering the discrepancy between 'in vitro' measured enzyme
activityingut contents and apparently (calculated) required enzyme activityfor digestion
of ingested feed components. With regard to the timing of enzymatic hydrolysis various
aspects of digestion are crucial, such as passage rate, mixing intensity,water content and
pH of the chyme.
Furthermore, the literature search (Chapter III) revealed that no consistency existswith
respect to methods ofsample collection and analysisof pancreatic (proteolytic) enzymes.
Enzyme activity depends on buffer composition, temperature, pH and substrate used.
These analytical factors differ greatly among studies resulting in poor comparability of
literature data.
An experiment was undertaken to study the effects of storage conditions (time and
temperature) on pancreatic enzyme activities (Chapter IV).From the results of this and
a follow-up experiment (Van Baak etal, 1991) it was decided to freeze-dry samples for
analysis of enzyme activities and store them at -20°Cbefore analysis to avoid reduction
inenzyme activities.These measures, however, donot solvetheproblem of comparibility
ofresults obtained bydifferent methods of analysis.Absolute values for enzyme activities
should be interpreted carefully but effects of specific treatments can be compared
adequately between experiments.
From the literature review (Chapter III) it was derived that a decline in pancreatic
enzyme activities often occurs after weaning. No detailed information isavailable on the
relation between dietaryprotein source,post-weaningfeed intakeand proteolytic enzyme
activities inyoung pigs.Therefore, twoexperiments were carried out to investigate these
relationships.
3.
Development ofpancreatictrypsinandchymotrypsinactivityafterweaninginrelation
to dietaryproteinsource
In Chapter V and VI two experiments have been described dealing with the effect of
dietary protein source on the development of protein digestive capacity inyoung piglets.
In both experiments, pancreatic trypsin and chymotrypsin activities have been measured
in both pancreatic tissue and small intestinal chyme. Pancreatic tissue samples were
pretreated with enterokinase to activate the precursors of trypsin and chymotrypsin
before measuring enzyme activities. Enzyme activities in tissue samples do not
necessarily correlate with enzyme activities at the site of luminal digestion (Pekas et al,
1964).
137
Chapter VIII
In the experiments described in Chapter V and VI positive correlations were found
between pancreatic and jejunal trypsin and chymotrypsin activities (Table 1).
Table 1.
Correlations between pancreatic tissue enzyme activities and jejunal chyme
enzyme activities (from experiments described in Chapter VandVI).
trypsin activity
(units per gram)
trypsin activity
(total units)
chymotrypsin activity
(units per gram)
chymotrypsin activity
(total units)
Chapter V
r
P
n
Chapter VI
r
P
n
0.487
0.0001
68
0.333
0.0052
69
0.477
0.0001
67
0.307
0.0103
69
0.262
0.0306
68
0.273
0.0230
69
0.091
0.4660
67
-0.217
0.0738
69
From Table 1 it can be seen that correlations between pancreatic and jejunal activity
werehigherand more significant for trypsin than for chymotrypsin.Thismayindicate that
the kinetics of intestinal (auto)hydrolysis of digestive enzymes are not the same for
trypsin as for chymotrypsin. The kinetics of pancreatic enzymes in the gut lumen with
respect to adsorption to digesta constituents, breakdown and lossof activity are not fully
known.
The production (synthesis + secretion) of trypsin and chymotrypsin by the pancreas
seems tobe regulated independently of each other aswaspreviously concluded bySnook
and Meyer (1964) from experimentswith rats and by Rothman (1976) from experiments
with rabbits.It canbe derived that thisprobably reflects the mode of pancreatic protease
synthesis and secretion. The regulation of pancreatic secretion is mediated through the
hydrolysis products of specific nutrients. Amino acids situated at specific cleavage sites
are triggers for the release of the corresponding protease from isolated rat pancreatic
tissue in vitro (Niederau et at, 1986). It was shown by Niederau et at (1986) that
digestive end products directly act on rat pancreatic zymogen granules, causing selective
release of specific digestive enzymes. Solomon (1987) summarized recent investigations
on the effects of intra-intestinal amino acids, peptides and proteins on pancreatic
secretion in different species. He concluded that intestinal (duodenum and jejunum)
peptidesand aminoacids,butnotintactproteins,stimulated pancreaticenzyme secretion.
It is unclear which specific amino acids are the most potent stimulants of pancreatic
secretion in different animal species.
Solomon (1987) suggested that the presence of pancreatic proteolytic enzymes in (the
138
Chapter VIII
upper third of) the small intestine suppresses pancreatic enzyme secretion in rats,
humans, cows, chickens, mice, hamsters and pigs, but not in dogs.
In the experiment described in Chapter V skim milk powder and soya protein
concentrate were used as dietary protein sources. Another experiment (Chapter VI)
comprised four diets based on skim milk powder, soya protein concentrate, soyabean
meal and fish meal, respectively. The diets were balanced with respect to lactose since
lactose hasbeen reported to affect protein utilization inyoung piglets (Sewell and West,
1965).
In Chapters V and VI it was shown that dietary protein source during the early postweaning period affects trypsin and chymotrypsin activities in pancreatic tissue and
intestinal contents.This effect wasmost pronounced at day 6after weaning (Chapter V)
and at day 3 and 6 after weaning (Chapter VI). Dietary skim milk powder caused the
highest jejunal enzyme activity in both experiments. In Chapter V this was the case for
chymotrypsin while in Chapter VI this was more evident for trypsin activity than for
chymotrypsin activity. These differences are difficult to explain because conflicting
evidence arises from literature: Efird et al. (1982) found higher jejunal trypsin and
chymotrypsin activities inpiglets fed soya proteins compared to piglets fed milk proteins
at one week after weaning (28days of age),while Newport and Keal (1982) and Owsley
etal.(1986) found lowerjejunal chymotrypsin activitywith pigletsfed soya proteins than
with piglets fed milk proteins.
Piglets fed fish meal had low trypsin activities in pancreatic tissue andjejunal digesta at
day 3 and day 6 after weaning (Chapter VI). Chymotrypsin activities in pancreas and
jejunum of piglets fed fish meal were high at day 3 and low at day 6 compared to the
other protein sources (Chapter VI).Ternouth etal.(1975) found lowamounts of trypsin
and chymotrypsin secreted incalvesfed dietsbased onfish protein concentrate compared
to diets based on skim milk powder or soyabean flour.
Jenkins et al. (1980) measured the extent of hydrolysis of various proteins by different
digestive enzymes separately in vitroand they found that skim milk is hydrolyzed to the
largest extent by trypsin and chymotrypsin (68.1 and 51.8%, respectively). An isolated
soyabean protein concentrate was hydrolyzed for 55.5 and 50.0% by trypsin and
chymotrypsin, respectively. Fish protein concentrate was hydrolyzed for 46.2 and 21.2%
by trypsin and chymotrypsin, respectively (Jenkins etal, 1980).Thiswould mean that to
reach a fixed level of protein hydrolysis more trypsin and chymotrypsin is needed with
fish and soya proteins than with skim milk protein. However, in our experiments
(Chapters V and VI) we found lower jejunal enzyme activities with piglets fed soya
proteins or fish meal (Table 2). From this we would expect a much higher protein
digestibilitywhen SMPwas fed due to higher amounts of proteolytic enzymes present in
the gut and due to higher susceptibility of milk proteins to hydrolysis by trypsin and
139
Chapter VIII
chymotrypsin. From Chapter II it can be seen that apparent ileal N digestibilities are
indeed lower when non-milk proteins are fed (Table 2).These differences are, however,
hardly reflected in true ileal N digestibility. The piglets used for the digestibility study
(Chapter II) were 2 to 3 weeks older than the piglets used for protease measurements
(Chapters V and VI).The differences inenzyme activitiesbetween newlyweaned piglets
fed different protein sources are not inagreement with the differences in (true) nitrogen
digestibility found with older piglets (Chapter II). This may indicate that the effect of
dietary protein source on jejunal protease activities diminishes or even reverses with
increasing age of the piglets.Thishypothesis issupported bythe results from Chapter VI
(Table 2) showing that at 3 days after weaning, the non-milk dietary proteins caused
significantly lower trypsin activityinthejejunum while these differences had disappeared
by day 6 after weaning.
From Chapters V and VI it is concluded that dietary protein source affects the activity
of trypsin and chymotrypsin in pancreatic tissue and jejunal digesta. The differences in
enzyme activities, however, cannot explain the differences in endogenous N losses as
found in Chapter II.
It must be stressed here, that digestibility figures from the 15N-isotope dilution
experiment (Chapter II) were obtained with piglets of about 7weeks old,who had been
fed the experimental diets for approximately 3 weeks before sample collection. These
piglets were therefore probably fully adapted to the diet composition and may be not
directly comparable with the newly weaned piglets used in the slaughter experiments
(Chapter V and VI).The differences between dietswith respect to endogenous nitrogen
losses as observed in the 15N experiment (Chapter II) probably do not fully reflect the
situation in newly weaned piglets.
During adaptation toanewdiet,digestive responses (passagerate,pH, enzymeactivities,
gut wall morphology, apparent and true digestibilities) are probably more pronounced
than in the case of fully adapted animals. In fully adapted piglets, the stressful event of
weaning is vanquished and the digestive system has adjusted to the new diet.
If the pancreas would be an important source of endogenous nitrogen secretion, this
should be reflected in the activity of the most important digestive enzymes secreted
(trypsin and chymotrypsin). One would then expect to find lower trypsin and
chymotrypsin activities injejunal chyme of piglets fed diets based on skim-milk powder
sincelowest endogenous ilealnitrogen losseswere observed when SMPwasfed (Chapter
II).
However, from Chapter V and VI it is concluded that feeding the SMP diet to newly
weaned piglets resulted in higher intestinal activities of pancreatic enzymes. Therefore,
itisconcluded that another source of endogenous Nsecretion isprobably responsible for
the extra endogenous N losses when non-milk proteins are fed to young piglets.
140
Chapter VIII
Table 2.
Summary of results obtained from Chapter II (1BN experiment, 4 weeks after
weaning)andChapter VI(first 10daysafter weaning).Theresultsareexpressed
as a percentage of the values obtained with piglets fed skim milk powder
(SMP=100%). SMP= skim milk powder, SI = soya protein isolate, SPC =soya
protein concentrate, SBM=soyabean meal,FM=fish meal.
Chanter II
apparent ileal N digestibility
true ileal N digestibility
ileal endogenous N loss
SMP
SI
100
100
100
93
106
251
ChaDter VI
feed intake day 0-3
100
feed intake day 3-6
100
feed intake day 6-10
100
relative pancreas weight at day 3
100
relative pancreas weight at day 6
100
relative pancreas weight at day 10
100
gastric pH at day 3
100
gastric pH at day 6
100
gastric pH at day 10
100
gastric pp/cp ratio day 3
100
gastric pp/cp ratio day 6
100
gastric pp/cp ratio day 10
100
total jejunal trypsin activity day 3
100
total jejunal chymotrypsin activity day 3
*
total jejunal trypsin activity day 6
100
total jejunal chymotrypsin activity day 6
100
total jejunal trypsin activity day 10
100
total jejunal chymotrypsin activity day 10100
SPC SBM
-
-
143
111
115
108
108
119
78
121
73
147
206
143
37
FM
remarks
91
98
181
86
96
198
SMP>SBM,FM
SI>SMP
SI>SMP
170
110
106
83
101
99
73
119
92
95
147
105
24
200
119
128
104
87
92
100
142
124
100
171
68
23
SBM,FM>SMP
NS
NS
SPC,FM>SBM
NS
SPC>SBM,FM
NS
NS
FM>SPC,SBM
NS
NS
SPC>FM
SMP>rest
SMP>FM
NS
NS
NS
NS
•
*
*
146
105
92
73
100
104
89
97
88
63
44
49
*
calculation of percentages not possibledu<
»to interaction between protein sourc
4.
Development ofpancreatictrypsin andchymotrypsinactivityafterweaninginrelation
topost-weaningfeed intake
i and feed intake.
From the results of the experiments described inChapters V and VI it also appears that
feed intake isan important factor in the'digestive development of newlyweaned piglets.
A positive relation was found between feed intake during the first sixdays post-weaning
and trypsin and chymotrypsin activities in pancreatic tissue and jejunal digesta. Braude
et al. (1970) reported a positive effect of increasing feed intake on jejunal proteolytic
enzyme activity in young piglets fed cow's milk. Lindemann et al. (1986) and Owsley et
al. (1986) suggested that the lowintestinal digestive enzyme activities they observed with
newly weaned piglets could be due to the depressed feed intake during the early postweaning period.
141
Chapter VIII
Feed intake of newlyweaned piglets isdifficult to control experimentally because of the
large variation in 'appetite' between individuals. Kelly et al. (1991a,b) used intragastric
gavage feeding to obtain controlled and continuous feed intake during the early postweaningperiod.Fromtheirstudiesonintestinalenzymedevelopment theyconcluded that
nutrient intake but also adaptational responses probably affect gut development. The
experiments reported by Kelly et al. (1991a,b) are of interest since they describe the
effects of feed intake on digestive adaptation during the early post-weaning period.
However, they applied tube feeding to control feed intake and this may have interfered
with 'normal' post-weaning development. To our knowledge, no experiments have been
described inliteraturewhere feed consumption duringthe earlypost-weaning periodwas
controlled in a more physiological manner.
In our experiments described in Chapters V and VI it was shown that feed intake is a
major factor in the development of digestive capacity ofyoungpigs.Piglets characterized
as 'non-eaters' during the first three post-weaning days (Chapter VI) demonstrated their
capacity of enzyme synthesis by the high trypsin and chymotrypsin activity in the
pancreatic tissue.The enzyme secretion into the gutwas probably lower for 'non-eaters'
than for 'eaters', indicated by the lowjejunal enzyme activities.
At day 6 after weaning, the positive relation between post-weaning feed intake and
jejunal enzyme activities was most pronounced (Chapter V and VI). The changes in
pancreatic response towards feed intake during the early post-weaning period
demonstrate that three phases may be distinguished: during the first three days after
weaning, thepiglet is'in trouble', aswasalsoproposed byLe Dividich and Herpin (1992)
who approached the post-weaning critical period from an energy-related point of view.
The digestive system is trying to cope with the changes imposed upon it, feed intake is
veryvariable and no clear response mechanisms canbe identified. Between 3and 6days
after weaning, the piglet clearly responds to changes in feed intake: higher feed
consumption is related to higher jejunal (and pancreatic) enzyme activities and longer
villi.Adaptation to the post-weaning situation is taking place. After day 6, the variation
in feed intake diminishes and the relation between feed intake and enzyme activities
disappears. The piglets have vanquished the nutritional stress related to weaning.
The influence of feed intake on the secretory capacity of the pancreas will probably also
depend on the patterns ofpost-weaning feed intake.Thisaspect could not be statistically
confirmed in our studies due to thevariation in feed intake patterns between piglets and
the relatively low number of animals used per experimental group. Studies from
Kamphues (1987) and Rothert (1987)indicate that after afasting/feed withdrawal period
ofone daypiglets consumed approximately 50%oftheir 'normal' dailyfeed intake within
the first 2hours after re-alimentation. This temporarily increased feed intake resulted in
higher gastricdigesta weights,higher gastric pH and lower intestinal pH, possibly leading
142
Chapter VIII
to a reduction in enzymatic protein hydrolysis.
It must be stressed here that in our experiments no different levels of feed intake were
imposed upon the piglets. Differences in feed intake are differences in voluntary feed
intake and relationsbetween feed intakeand,e.g.,enzyme activitiesmaytherefore reflect
differences in 'adaptational status' of the piglets rather than causal effects of feed intake
per se.
The actual secretion of trypsin and chymotrypsin into the gut lumen seems to be
triggered by the presence of food (substrate) in the digestive tract (Chapter VI). From
Chapter VI it can be concluded that feed intake and dietary protein source may interact
withrespect topancreatic enzymesynthesisand secretion.Aregular and gradual increase
infeed intake after weaningisprobablyimportant for anundisturbed development ofthe
pancreatic secretory capacity in young piglets.
Allpigletssacrificed at 6daysafter weaning had abnormal gutwallmorphology (Chapter
VI) with respect to villus shape. No gut wall samples were taken at weaning, but it may
be expected from literature data (Chapter VI) that the gut wall damage was due to
weaning. The decrease in relative small intestinal weight after weaning (Chapter VI) is
in accordance with this finding.
The positive relation between post-weaning feed intake and jejunal villus lengths and
crypt depths may indicate a stimulatory effect of feed intake on gut wall maturation
(Chapter VI). Kelly et al. (1991b) also found a relationship between feed intake
(controlled bytube feeding) and villus length and crypt depth at different sites along the
small intestine.Thisfinding merits further study,sincevillusatrophy isa common finding
inweaned piglets and gutwalldamage maycontribute to increased endogenous nitrogen
losses and to decreased absorptive capacity of the intestinal mucosa.
From the evidence presented in Chapters V and VI it is concluded that post-weaning
feed intake hasa greater influence on the development ofpost-weaning protein digestive
capacity than dietary protein sourceper se.
Experimental evidence for the crucial role of feed intake (patterns and quantity) during
the early post-weaning period is still lacking. Because voluntary feed intake in newly
weaned pigletsisgenerallylow,irregular and veryvariable,itisdifficult to study different
feeding strategies with these animals. Forced feeding imposes an additional stress factor
upon the newlyweaned piglet, which will interfere with the already stressed state of the
young pig.
The regulation of feed intake, especially in newly weaned piglets is insufficiently
understood and requires further study.
143
Chapter VIII
5.
Gastricprotein breakdown, digestive tractpH and development ofprotein digestive
capacity
The ratio between TCA-precipitable protein and crude protein (pp/cp ratio) measures
the relative amount of non-hydrolyzed protein. The pp/cp ratio in gastric digesta at
weaning was similar in the two experiments described (0.67 in Chapter V and 0.64 in
Chapter VI). These piglets had only received sows milk (no solid feed) and the results
indicate that the degree of gastric protein breakdown (reciprocal of pp/cp) was
approximately 35 % when piglets were suckling the sow.
With regard to gastric protein breakdown after weaning, different results were obtained
in the two experiments. In the first experiment (Chapter V) a clear effect of dietary
protein source on gastric protein breakdown was found. About 45 % of the protein from
skim milk powder was hydrolyzed in the stomach, while only 1to 14 % of the protein
from soya protein concentrate was broken down in the stomach. This intriguing result
could not be reproduced in the second experiment (Chapter VI).
The degree of gastric protein breakdown at day 3 and day 6 after weaning was much
higher in the second experiment (65 to 83 %, Chapter VI) than in the first experiment
(1to 44 %, Chapter V).Only at day 10after weaning an effect of dietary protein source
was found (Chapter VI): dietary fish meal resulted in a higher degree of gastric protein
hydrolysis than soya protein concentrate. The method of analysis of TCA-precipitable
protein was the same in both experiments and the pp/cp ratio's at weaning are similar
for both experiments (as expected). The composition of the diets and the time between
feeding and slaughter were similar for the two experiments described in Chapter V and
VI. The differences in post-weaning gastric protein breakdown between the two
experiments may be related to age of the piglets at weaning (25 days in Chapter V and
28days in Chapter VI),gastric pH (higher in Chapter VI) and/or feed intake during the
first three dayspost-weaning (175 g/din Chapter Vand 56-112 g/d in Chapter VI). The
difference in weaning age between the two experiments was only three days and
therefore it is unlikely that this would result in a large difference in gastric protein
breakdown. From the lowgastric pH (closer to the pH optimum of pepsin) found in the
first experiment (Chapter V) compared to the second experiment (Chapter VI) a higher
degree ofprotein breakdownwouldbe expected inthe first experiment (Chapter V).The
higher initial feed intake in Chapter V, resulting in higher gastric digesta weight at day
3might have inhibited gastric protein breakdown. Itwas not possible to studyinitial feed
intake in Chapter V in more detail. It is possible that piglets had irregular feed intake
patterns leading to disturbances in gastric protein breakdown, possibly related to gastric
emptying.
The pH of the gastricdigesta inboth experimentswashigher than the optimum pH value
144
Chapter VIII
for pepsin action. Gastric pH was higher than values reported by Hartman etal. (1961)
but agreed with data from Efird et al. (1982). Maner et al. (1962) found lower intragastric pH with four weeks old piglets fed liquid diets based on casein than with piglets
fed liquid diets based on isolated soya protein (3.2 vs 5.0) measured one hour after
feeding. These contradictory results may be due to the form of the feed (liquid vs dry).
Maner etal. (1962) found in addition a difference in total gastrointestinal tract passage
rate between the two diets at four weeks of age (19 hours for the isolated soya protein
dietvs45hoursfor the casein diet).Thisdifference had disappeared at tenweeks of age.
In Figure 1the influence of buffer pH on trypsin and chymotrypsin activity is illustrated.
In our experiments, the pH in the small intestine of newly weaned piglets (Chapter V
and VI)was 1to 3unitslower than the pH optimum for trypsin and chymotrypsin action.
Therefore, trypsin and chymotrypsin activity 'in situ' (Chapter V) were probably only 12
to 25% of the activity measured 'in vitro'. In the experiment described in Chapter VI,
jejunal pH ranged from 6.8 to 7.8 and therefore the 'effective' enzyme activity was
probably 15 to 45% for trypsin and 40 to 85% for chymotrypsin (Figure 1).The pH in
the intestinal digesta can be of major importance for the 'expression' of enzyme activity
'in vivo'.
6.
Can adeficiencyinpancreaticproteolyticenzymesynthesis/secretion bethe causeof
poorpost-weaningpigletperformance?
During the early post-weaning period piglet performance isoften impaired. Manypiglets
display irregular feed intake: during the first few hours or days after weaning no solid
feed is ingested, while afterwards hunger strikes and piglets tend to overeat. This
phenomenon may be an important cause of post-weaning disorders. The gradual
adaptation of the piglet's immature digestive system is impaired with respect to motility
(Sissons, 1989),enzyme secretion (Jones, 1986)and pH regulation (Bolduan etal.,1988).
The research described in this thesis was (among other things) aimed at studying the
possibility that a lack of pancreatic proteolytic enzyme synthesis and/or secretion could
be a cause for depressed digestion and performance in newlyweaned piglets.Studies by
Lindemann et al. (1986) and Owsley et al. (1986) have shown depressed pancreatic
enzyme activities in pancreatic tissue and intestinal chyme of piglets during the first few
days after weaning.
Post-weaning feed intake is expected to be crucial in this respect because of the
importance of substrate availability for the development of pancreatic secretion.
Newly weaned piglets are capable of synthetizing and secreting appreciable amount of
pancreatic proteases in response to dietary stimuli (feed intake and dietary protein
145
Chapter VIII
source).InChapter VIIitisshownthat evenbefore weaning pigletsrespond to hormonal
stimulation with an increase in pancreatic secretion (volume and enzyme activities) (see
also Harada et al, 1986, 1988).
The relation between feed intake during the first three days post-weaning and enzyme
activitieswas not significant (Chapter V). From Chapter VI it can be derived that when
feed intake during the first three days post-weaning iszero or close to zero, trypsin and
chymotrypsin activities in pancreatic tissue are high, indicating a distinct capacity to
synthetize these enzymes immediately after weaning (Chapter VI).
This capacity also arises from results presented in Chapter VII. Especially trypsin
secretion (units per hour) increased in young piglets when a hydrolyzed casein solution
ofpH 2.0wasinfused intothe duodenum.Harada etal.(1986)found a dramatic increase
of pancreatic juice flow and protein output in young anaesthetized piglets after
intraduodenal infusion of a HC1 solution with pH less than 1.5. Above this pH value
insufficient secretin was released to induce increased secretion. The two- to five-fold
increase injuice volume secreted as reported in Chapter VII may be the result of both
low intraduodenal pH and the presence of products of protein breakdown (hydrolyzed
casein).
Harada etal.(1988) showed that 21-day-oldpigletsresponded toan intravenous secretin
injection with a four- to 15-fold increase inpancreaticjuice secretion. From the evidence
presented byHarada etal. (1986, 1988) and in Chapter VII it is clear that young piglets
are capable of increasing their pancreatic secretion in response to intravenous and
intraduodenal stimuli.
A schematic representation of digestive eventsoccurring atweaning
Stress may be defined as tension or pressure caused by environmental changes and
requiring adaptational action from the animal. Typical physiological and biochemical
responses of the animal to a stressor include changes in gastric secretion and motility,
increases in heart rate and blood pressure, and increases in the levels of plasma
corticosterone, Cortisol and catecholamines (Gue et al, 1989). When the environmental
change (e.g., weaning) comprises a severe strain to the animal, adaptation may be
difficult or even impossible and acute stress may shift towards chronic stress, especially
when coping strategies employed by the animal turn out to provide non-satisfactory
results.
Although 'stress' is an ill-defined process, it is generally accepted (e.g. Stanton and
Mueller, 1976; Worsaae and Schmidt, 1980) that early weaning is a stressful event
comprising acute and chronic aspects: many factors in the environment of the young
146
Chapter VIII
animal undergo a sudden change, e.g., the piglets are removed from their dam, the
piglets are often transferred to another pen, the piglets are presented with another type
and acquisition of food (dryvsliquid, hard vssoft, cold vswarm, eatingvssuckling).The
young piglet has to cope with these changes appropriately. Generally stress invokes
physiological responses involving hormonal, neural and behavioural mechanisms. Stress
related hormones like ACTH (adrenocorticotropic hormone), corticosteroids and
catecholamines (adrenalin and noradrenalin) play a key role in the response to various
stressors (Gue et ah, 1989; Dantzer and Mormede, 1983).
Weaning comprises stressto theyoungpiglet and itmaytherefore be ofinterest to study
the effect of stress-related hormones on digestive development of newlyweaned piglets.
This was done by Chappie et ah (1990a,b,c).
Chappie et ah (1990a,b,c) concluded from their studies on the effects of ACTH and
glucocorticoids on newlyweaned pigperformance, that thesehormones can stimulate the
development of the pancreatic and intestinal enzyme system.
They found that glucocorticoid administration to nursing piglets can evoke premature
elevation of the pancreatic and intestinal carbohydrase enzymes either through a direct
effect on digestive capacity or indirectly through stimulation of feed intake.
Stress increases the need for energy and enhances general wakefulness (Uvnas-Moberg,
1989).
Fromthefield ofanimalethologyinterestingmechanisms havebeen proposed (e.g.Selye,
1956; Dunn and Kramarcy, 1984) concerning adaptation to stress factors and coping
strategies of animals.
It is proposed that analogous mechanisms may be applied to the digestive (nutritional)
adaptation of piglets during the early post-weaning period. A schematic representation
of possible coping mechanisms of the young pig in response to the nutritional aspects of
the post-weaning situation ispresented in Figure 2.The piglet mayrespond to itsgeneral
state of stress by refraining from eating. Thiswas seen in our experiments (Chapter VI)
with 33% of the piglets fed the skim milk powder diet, 20% of the piglets fed the soya
protein concentrate diet, 13%of the piglets fed the soyabean meal diet and none of the
piglets fed the fish meal diet during the first three post-weaning days.
This 'coping strategy' can bring about various physiological consequences: the
gastrointestinal tract adapts to the fasting state by changing its motility and secretion
patterns (Sissons, 1989).The piglet may lose weight and find itself in a negative energy
balance which will increase its susceptibility to infections and diseases. After fasting for
some hours or days,the pigletwillget hungry and start eating.Thiswillupset itsdigestive
system (Kamphues, 1987) and may lead to severe digestive disorders (diarrhoea).
The critical period of underfeeding during the first days after weaning was also
recognized by Le Dividich and Herpin (1992), who focused on the negative energfy
147
Chapter VIII
balance of newly weaned piglets.
WEANING
(stressor)
GRADUAL INCREASE
IN FEED INTAKE
FASTING
Fl
Fl
days
days
HUNGER
OVEREATING
days
SATISFACTORY
PERFORMANCE
DIGESTIVE
DISORDERS
Figure2.
General mechanism of coping strategies in response to a nutritional stressor
(weaning) in youngpiglets.
When early post-weaning feed intake is not impaired, the piglet gradually increases its
feed intake after weaning and adapts its digestive system to the new feeding system and
feed composition. Thisrequires an adaptation of the different digestive organs (stomach,
pancreas, small intestine) with respect to motility (gastric emptying, intestinal passage
148
Chapter VIII
rate), pH regulation (stomach pH, pancreatic and intestinal secretion),gutwall integrity
(villus length and crypt depth) and digestive enzyme synthesis and secretion (stomach,
pancreas, small intestine). As long as the piglet is not experiencing too severe stress,
these adaptations may occur relatively smoothly. However, the demands imposed upon
the piglet's digestive system may be too large when diets of a new composition and
structure are fed. The familiarity of form and composition of the post-weaning diet may
play an important role in this respect. An unfamiliar feed or feeding method may
provoke inappropriate responses of the piglet's digestive system.
A complicating factor in the case of nutrition of newlyweaned piglets is that the uptake
of nutrients into the organism is disturbed resulting in loss of condition and increased
susceptibility towards stress and disease.
Conclusions
In young piglets at four weeks after weaning, the differences in apparent digestibility
between skimmilkpowder and soyaproducts and fish meal are mainlydue to differences
in endogenous nitrogen losses and much less to differences in true nitrogen digestibility.
The differences in endogenous nitrogen losses do not correspond with the differences in
pancreatic protease activities in newly weaned piglets, indicating that the major source
of endogenous nitrogen loss is not the pancreas.
Pancreatic enzyme activities as measured in jejunal digesta are not elevated in piglets
losing high amounts of endogenous nitrogen at the terminal ileum. This indicates that
pancreatic proteases are not the main source of endogenous nitrogen when diets based
on skim milk powder, fish meal or soya proteins are fed. Other sources of endogenous
nitrogen are probably more important and gut wall damage may be a critical factor in
this respect.
From the experimental results presented in this thesis it is concluded that pancreatic
enzyme production as such is not the 'bottle neck' in newlyweaned piglet performance.
Even before weaning, piglets are quite capable of adapting their enzyme secretion when
an appropriate stimulus isgiven.The post-weaning decline in enzyme activities as found
by Efird et al. (1982), Lindemann et al. (1986) and Owsley et al. (1986) is most likely
related to poor feed intake after weaning.
In our studies we demonstrated that feed intake during the early post-weaning period is
more important than dietary protein source for the development of the piglet's digestive
system. The feed intake of newly weaned piglets may be stimulated by feeding diets of
appropriate composition as was shown in our experiments for the piglets fed the fish
meal diet.
149
Chapter VIII
The detrimental effects of fasting during the first day(s) after weaning are possibly
mediated through an increased gastrointestinal motility during fasting, leading to an
increased digesta passage rate when the piglets start consuming the post-weaning diet.
This phenomenon could hamper digestion and provoke bacterial growth, fermentation
and gastrointestinal disorders (Sissons, 1989).
The dramatic lack of appetite in newly weaned piglets may be described to
environmental, social ordietary stressfactors correspondingwith the process ofweaning.
The chemical and physical properties of the feed might be of importance when
attempting to stimulate agradual increase in feed intake of newlyweaned piglets.In our
studies fish meal resulted in increased feed intake of young piglets (Chapter VI) aswas
also found by Newport and Keal (1983).
Recommendations for further research
The identification and quantification of the various sources of endogenous nitrogen and
the dietary factors influencing these sources should be an important issue in future
research.
The regulation of feed intake infarm animals requires further study.Especially in newly
weaned piglets, the effects of 'stress' on feed intake and digestive development should
be examined in detail. Patterns of feed intake during the first week post-weaning should
be studied in more detail.
The interrelationships of gastric and pancreatic functioning during digestive adaptation
should be investigated in more detail: Research on isolated organs may provide
knowledge on separate regulation mechanisms, but integrated responses to (nutritional)
stimuli are of greater importance.
The regulation of exocrine pancreatic secretion merits further study, since contradictory
results concerning the effects of weaning and diet composition are obtained from
different studies. The different methods of analysis of (pancreatic) digestive enzymes
should be compared and evaluated to detect artefact literature data arising from
erroneous sample storage or analysis.The relation between enzyme activities measured
using specific substrates and optimum assay conditions and enzyme activities actually
expressed in the digestive tract is completely unclear and certainly merits further study.
The differences in pancreatic enzyme secretion when different diets are fed are difficult
to interpret because it is not completely known how much enzyme activity is needed to
digest a certain amount of dietary protein. Therefore, one cannot decide conclusively if
there is either an excess or a shortage of digestive enzymes secreted by the pancreas.
150
Chapter VIII
Implications
scientific
The digestion of protein is a complex process accomplished by the combined actions of
the stomach, the pancreas and the small intestine. The regulation of pH in the digestive
tract (stomach andsmallintestine)and thesynthesisandsecretion ofproteolytic enzymes
(stomach,pancreas and smallintestine) control the digestion of dietaryprotein.An intact
small intestinal wall is essential for the absorption of the products of protein digestion.
From the research described inthis thesis it isconcluded that differences in endogenous
nitrogen losses are the primary cause of differences indigestion between protein sources
in young piglets. The origin of this endogenous nitrogen is insufficiently clear yet. The
pancreas certainlyplaysanimportant roleinthedigestion ofprotein,but isnotthe major
source of endogenous nitrogen lossesinyoung piglets fed diets based on soya protein or
fish meal.The importance ofpost-weaningfeed intake (patterns) isclearly demonstrated
inthisstudy.Interactionsbetween animalfactors (adaptation after weaning,feed intake)
and dietary factors (dietary protein source) were eminent in this study and stress the
importance of an integrated approach in the study of digestive processes.
practical
The study of factors affecting protein digestion in farm animals isvital for improving the
efficiency of animal production. Optimizing protein digestion will reduce the costs of
animal production and the excretion of excessive amounts of minerals.The performance
of piglets during the early post-weaning period is still unsatisfactory.
The research described in this thesis demonstrates that feed intake and dietary protein
source are important factors in post-weaning pig performance. Pancreatic enzyme
secretionperse is probably not a limiting factor in protein digestion, since young piglets
are able to respond to stimuli with an increased secretion of digestive enzymes into the
digestive tract. It is therefore not advisable to supplement weaner diets with exogenous
proteolytic enzymes. Stimulation of endogenous enzyme secretion may, however, be
insufficient in the newly weaned piglet. It is recommended to promote the piglet's
indigenous capacity to synthetize and secrete digestive enzymes around weaning. This
may be done by avoiding sudden changes at weaning. Minimizing the stress of weaning
may help to stimulate feed intake during the early post-weaning period. Piglets should
be encouraged to start feed consumption immediately after weaning.A post-weaning lag
in feed intake lasting for more than a few hours should be prevented. The composition
of the diet plays an important role herein.
151
Chapter VIII
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infusion ofhydrochloric acid and monocarboxylic acidin anesthetized piglets.Jap.
J.Physiol. 36:843-856
Hartman, P.A., Hays, V.W., Baker, R.O., Neagle, L.H., Catron, D.V., 1961.
Digestive enzyme development in the young pig./. Anim. Sci. 20:114-123
Hee, J.H., 1984.
Pancreatic Secretions in the GrowingPig.Thesis. UniversityofAlberta, Edmonton,
Canada
Huisman, J., 1990.
Antinutritional Effects of Legume Seeds in Piglets, Rats and Chickens. Thesis.
Agricultural University, Wageningen, TheNetherlands
Jenkins, K.J., Mahadevan, S., Emmons, D.B., 1980.
Susceptibility of proteins used in calf milk replacers to hydrolysis by various
proteolytic enzymes. Can.J.Anim. Sci. 60:907-914
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Jones, E.E., 1986.
Biological development and nutritional requirement of the neonatal pig.
Proceedings of the Georgia Nutrition Conference1986. pp 145-162
Just, A., J0rgensen, H., Fernandez, J.A., 1981.
The digestivecapacityofthe caecum-colon andthevalue ofthe nitrogen absorbed
from the hind gut for protein synthesis in pigs.Br.J. Nutr. 46:209-219
Kamphues, J., 1987.
Untersuchungen zuVerdauungsvorgangen beiAbsetzferkeln inAbhangigkeit von
Futtermenge und -zubereitung sowie Futterzusatzen. Thesis. Tierarztliche
Hochschule Hannover, Hannover, Germany
Kelly, D., Smyth, J.A., McCracken, K.J., 1991a.
Digestive development of the early-weaned pig. 1. Effect of continuous nutrient
supply on the development of the digestive tract and on changes in digestive
enzyme activity during the first week post-weaning.Br.J.Nutr. 65:169-180
Kelly, D., Smyth, J.A., McCracken, K.J., 1991b.
Digestive development of the early-weaned pig. 2. Effect of level of food intake
ondigestiveenzymeactivityduringtheimmediate post-weaningperiod,fir./. Nutr.
65:181-188
Lindemann, M.D.,Cornelius, S.G., ElKandelgy,S.M., Moser, R.L.,Pettigrew,J.E., 1986.
Effect of age,weaning and diet on digestive enzyme levels in the piglet./. Anim.
Sci. 62:1298-1307
Maner, J.H., Pond, W.G., Loosli, J.K., Lowrey, R.S., 1962.
Effect of isolated soybean protein and casein on the gastric pH and rate of
passage of food residues in baby pigs.J. Anim. Sci. 21:49-52
Newport, M.J., Keal, H.D., 1982.
Artificial rearing of pigs. 12.Effect of replacement of dried skim-milk by either
a soya-protein isolate or concentrate onthe performance of the pigsand digestion
of protein. Br.J.Nutr. 48:89-96
Newport, M.J., Keal, H.D., 1983.
Effect of protein source on performance and nitrogen metabolism of pigsweaned
at 21 days of age.Anim. Prod. 37:395-400
Niederau, C , Grendell, J.H., Rothman, S.S., 1986.
Digestiveend productsrelease pancreaticenzymesfrom particulate cellularpools,
particularly zymogen granules.Biochim. Biophys. Acta 881:281-291
Owsley, W.F., Orr, D.E., Tribble, L.F., 1986.
Effects of age and diet on the development of the pancreas and the synthesis and
secretion of pancreatic enzymes in the young pig./. Anim. Sci. 63:497-504
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Pekas, J.C., Hays, V.W., Thompson, A.M., 1964.
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Rothert, J., 1987.
Veranderungen derChymuszusammensetzungbeiAbsetzferkeln nach iiberhohter
Futteraufnahme. Thesis.TierarztlicheHochschuleHannover, Hannover, Germany
Rothman, S.S., 1976.
Independent secretion of different digestive enzymes by the pancreas. Am. J.
Physiol. 231(6):1847-1851
Schulze, H., Makkink, C.A., Leeuwen, P.van, Verstegen, M.W.A., 1993.
The effect ofthe level ofisotope infusion and the choice ofprecursor poolon the
determination of endogenous ileal nitrogen excretion in the young pig using the
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Some effects oflactose on protein utilization inthe baby pig./.Anim. Sci. 24:239241
Sissons, J.W., 1989.
Aetiology of diarrhoea in pigs and pre-ruminant calves. Chapter 14 in: Recent
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Response of digestive enzymes to dietary protein./. Nutr.82:409-414
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Control of exocrine pancreatic secretion. Chapter 43 in: Physiology of the
GastrointestinalTract(SecondEdition). Volume2.EdsL.R Johnson,J. Christensen,
MJ. Jackson,E.D.Jacobson,J.H.Walsh;RavenPress, NewYork,USA.ppl173-1207
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1989:60-63?
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Digestion in the pig between 7 and 35 days of age. 3.The digestion of nitrogen
in pigs given milk and soyabean protein. Br.J.Nutr. 45:337-346
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156
SUMMARY
157
Summary
Introduction
Over the past decades piglet weaning age has decreased from months to three to four
weeks.With thisearlyweaning digestive disorders innewlyweaned pigletshave become
more apparent. Especially protein digestion depends on ageofthe pig:youngpiglets are
less capable of digesting protein sources of vegetable origin. The difference in
digestibility between animal and plant protein sources diminishes with advancing ageof
the piglets (Chapter I).
The causes of the lowdigestibility of certain protein sources innewlyweaned piglets are
not fully understood. Some authors have claimed that the secretion of (pancreatic)
enzymes isinsufficient innewlyweaned piglets,causing depressed protein digestion and
sometimes digestive disorders (diarrhoea) in young piglets.
The main objective of thepresent investigationswastostudythedevelopment of protein
digestive capacity in young weaned piglets.
Effect of dietaryprotein sourceon endogenousnitrogen lossesinyoungpigs
To study the digestion of protein in detail, an experiment was carried out to determine
the endogenous nitrogen losses in young piglets when different dietary protein sources
werefed (Chapter II).Itwasconcluded that differences inapparent nitrogen digestibility
between skim milk powder, soya proteins and fish meal were mainly due to differences
inendogenous nitrogen losses.The differences intruenitrogen digestibilitywere smaller
than the differences in apparent nitrogen digestibility. Endogenous ileal nitrogen losses
were 2 to 2Vz times higher when non-milk protein were fed.
From thisfinding itwasdecided toinvestigate a possible source of endogenous nitrogen,
the exocrinepancreas.The pancreasisan important gland secretingproteolytic enzymes
and therefore a reviewwas made of literature on pancreatic secretion in pigs (Chapter
III). No consistency exists with respect to methods of sample collection and analysis of
(pancreatic) enzymes. Therefore, we conducted an experiment to study the effect of
storage conditions (time and temperature) on the activity of trypsin, chymotrypsin and
lipase in pancreaticjuice (Chapter IV).It was found that storage time and temperature
affect enzyme activities and therefore it was decided to freeze-dry samples for enzyme
analysis and store them frozen to minimize changes in enzyme activities.
Pancreatic secretion adapts to changes in dietary composition and often a decline in
pancreatic enzyme secretion isfound duringthe earlypost-weaning period. Research on
pancreatic enzyme secretion in newly weaned piglets usually focuses on dietary
composition and weaning age. Post-weaning feed intake is never taken into account,
158
Summary
especially because piglets are usually not housed individually after weaning.
Because individual feed intake of newly weaned piglets is thought to be of major
importance for the development of pancreatic secretion, this aspect was taken into
account in two experiments.
Effect of dietaryprotein sourceandfeed intake
onpancreaticsecretion in newlyweanedpiglets
Two experiments comprising 70 piglets each were carried out to study trypsin and
chymotrypsin activities in pancreatic tissue and jejunal digesta of newly weaned piglets
fed dietsbased on either skim milk powder, soya protein concentrate, soyabean meal or
fish meal (Chapters V and VI). Feed intake of the piglets was recorded daily.
Trypsin and chymotrypsin activities in pancreas and jejunum were affected by dietary
protein source, especially during the first week after weaning. Skim milk powder
generallyresulted inhighjejunal protease activities.Fishmealcausedlowpancreatic and
jejunal trypsin activityduringthefirst post-weaningweekand highpancreaticand jejunal
chymotrypsin activities at day 3 after weaning. Pancreatic and jejunal chymotrypsin
activities were low at day 6 after weaning when fish meal was fed.
The influence of dietary protein source on trypsin and chymotrypsin activities was most
pronounced at day 3 after weaning and had disappeared at day 10.
The relation between feed intake and (jejunal) enzyme activities was more persistent:
The effect of feed intake seemed to be greater than the effect of dietary protein source.
It was concluded from Chapters V and VI that the first three days after weaning
comprise the mostdifficult period for theyoungpiglet.Many pigletsconsumed lessthan
10 grams feed per day and these 'non-eaters' had lowerjejunal enzyme activities than
piglets consuming more feed. Between 3and 6days after weaning, the relation between
feed intake and enzyme activities became clear. The piglets were adapting to the new
nutritional situation. By day 10, the differences in enzyme activities between diets had
disappeared, the piglets had overcome the post-weaning nutritional difficulties.
Capacityofyoungpigletsto respondto stimulation ofpancreatic secretion
A final experiment was performed to check the ability of the young piglet to respond to
pancreatic secretion stimulation (Chapter VII). It was shown that an increased
pancreaticsecretionwasobtainedwhensecretinwasinjected intravenously.Even before
weaning,pancreaticsecretion responded tointraduodenal infusion ofahydrolyzed casein
159
Summary
solution of pH 2.Thisfinding confirmed the idea that pancreatic secretion assuch isnot
the 1>otfle-neck' in newly weaned piglet performance. The disturbed feed intake
(quantity and pattern) often seen after weaning may,however, hamper smooth (protein)
digestive capacity development after weaning in young piglets.
More (practical and scientific) attention for feed intake (patterns) duringthe early postweaning period could provide tools for the improvement of post-weaning piglet
performance.
Conclusions
Differences in apparent nitrogen digestibility between skim milk powder, soya products
and fish meal in young piglets are due mainly to differences in endogenous nitrogen
losses.
The exocrine pancreas is not the major source of endogenous nitrogen losses in young
piglets.
Newly weaned piglets have the ability to respond to intravenous or intraduodenal
stimulation by increasing their pancreatic secretion.
Inyoungpiglets,post-weaningfeed intakeismoreimportant than dietaryprotein source
for the development of pancreatic protease activities.
Dietary composition may influence post-weaning feed intake in young piglets.
Insufficient consistencywithrespect tosample storage and enzyme analysishampers the
interpretation of literature data.
160
SAMENVATTING
161
Samenvatting
Inleiding
De speenleeftijd van biggen is over de laatste decennia teruggebracht van maanden tot
drie avierweken.Door ditvroege spenen zijn verterings-problemenbij pas-gespeende
biggen duidelijker naarvorengekomen.Vooral deeiwitverteringhangt afvande leeftijd
van de big:jonge biggen zijn minder goed in staat om plantaardige eiwitten te verteren
dan oudere biggen. Het verschil in verteerbaarheid tussen dierlijke en plantaardige
eiwitten neemt af met toenemende leeftijd van de big (Hoofdstuk I).
De oorzaken van de lageverteerbaarheid van bepaalde eiwitbronnen bij pas-gespeende
biggen zijn nog niet volledig duidelijk. Sommige onderzoekers beweren dat de secretie
van (pancreas-)enzymen onvoldoende isinpas-gespeendebiggen,wat danzou leiden tot
verminderde eiwitvertering en soms verterings-storingen (diarree) bij jonge biggen.
Debelangrijkste doelstellingvanhetonderhavigeonderzoekwasomdeontwikkelingvan
de eiwitverteringscapaciteit injonge biggen nader te bestuderen.
Effect van eiwitbronin het voerop de ilealeendogenestikstofverliezen injonge biggen
Om de eiwitvertering gedetailleerd te bestuderen werd een experiment uitgevoerd om
de endogene stikstof-verliezen vast te stellen bij jonge biggen, die voeders met
verschillende eiwitbronnen kregen (Hoofdstuk II).Uitderesultatenvandezeproef werd
geconcludeerd, datverschillen inschijnbare stikstof vertering tussen ondermelk poeder,
soja eiwitten en vismeel vooral te wijten waren aan verschillen in endogene stikstof
verliezen. De verschillen in werkelijke eiwitvertering waren kleiner dan de verschillen
in schijnbare eiwitvertering. De endogene ileale stikstof verliezen waren 2 tot 2'/2 keer
zo hoog bij voeders met soja of vismeel.
Hierna werd besloten een mogelijke bron van endogeen stikstof te onderzoeken,
namelijk de exocrine pancreas. De pancreas is een belangrijke klier, die proteolytische
enzymen uitscheidt en daarom werd een overzicht gemaakt van de literatuur met
betrekking tot pancreas-sacretie bijvarkens (Hoofdstuk III).Voor wat betreft de opslag
en analyse van monsters voor enzymbepalingen is er weinig overeenstemming in de
literatuur. Daarom werd een experiment opgezet om het effect vast te stellen van
bewaar-omstandigheden (tijdsduur en temperatuur) op de activiteit van trypsine,
chymotrypsine en lipase in pancreassap (Hoofdstuk IV). Er werd gevonden dat
bewaartijd en -temperatuur invloed hebben op de enzymactiviteiten. Daarom werd
besloten om monsters bestemd voor enzymanalyse te vriesdrogen en te bewaren bij -20
°C om verlies van activiteit te vermijden.
Pancreassecretie past zich aan aan veranderingen in voersamenstelling en vaak wordt
162
Samenvatting
een teruggang in enzymactiviteiten na het spenen gezien. Onderzoek naar de
enzymsecretie door de pancreas van pas-gespeende biggen is meestal gericht op
voersamenstelling en speenleeftijd. De voeropname na het spenen wordt vrijwel nooit
gemeten,waarschijnlijk omdatpas-gespeende biggenmeestal niet individueelgehuisvest
worden.
Omdat hetvermoeden bestaat, dat de individuele voeropname van de biggen van groot
belang voor de ontwikkelingvan de pancreassecretie, isdit aspect meegenomen in twee
proeven.
Effect van eiwitbronen voeropname oppancreassecretie bijpas-gespeende biggen
Twee experimenten, elk bestaande uit 70biggen,werden uitgevoerd om de trypsine- en
chymotrypsine-activiteit vast te stellen in pancreasweefsel en jejunumchymus van pasgespeende biggen. Vier verschillende rantsoenen werden samengesteld, gebaseerd op
ondermelkpoeder, soja-eiwit-concentraat, sojaschroot ofvismeel (Hoofdstuk V en VI).
De voeropname van de biggen werd dagelijks gemeten. Trypsine en chymotrypsine
activiteiten in pancreas enjejunum werden beinvloed door eiwitbron, vooral gedurende
de eerste week na spenen. Ondermelkpoeder leidde in het algemeen tot hoge protease
activiteiten inhetjejunum. Vismeelveroorzaakte een lagetrypsine activiteit in pancreas
en jejunum tijdens de eerste week na spenen en een hoge chymotrypsine activiteit in
pancreas en jejunum op dag 3 na spenen. Chymotrypsine activiteit in pancreas en
jejunum was laag op dag 6 na spenen als vismeel werd gevoerd.
Het effect van eiwitbron in het voer op de trypsine- en chymotrypsine-activiteiten was
het duidelijkst op dag 3 na spenen en was verdwenen op dag 10.
De relatie tussen voeropname en enzymactiviteiten (in het jejunum) was sterker: het
effect van voeropname leek groter te zijn dan het effect van eiwitbron. Uit Hoofdstuk
V en VIwerd geconcludeerd dat de eerste drie dagen na het spenen het moeilijkst zijn
voor de jonge big. Veel biggen aten minder dan 10gram per dag en deze 'niet-eters'
hadden lagere enzym-activiteiten in hetjejunum dan biggen die meer aten. Tussen dag
3en dag 6tekende zich de relatie tussen voeropname en enzymactiviteiten duidelijk af.
De biggen pasten zich aan aan de nieuwe voedings-omstandigheden. Op dag 10waren
de verschillen in enzymactiviteiten tussen voeders verdwenen, de biggen hadden de
(voedings)problemen overwonnen.
163
Samenvatting
Het vermogenvanjonge biggen om tereageren op stimulatie vanpancreas-secretie
Een laatste experiment werd gedaan om het vermogen van dejonge bigom te reageren
oppancreas-stimulatie tetesten (Hoofdstuk VII).Eentoenamevande pancreas-secretie
werd gevonden na intraveneuze secretine-injectie. Zelfs voor het spenen reageerde de
secretie op een intra-duodenale infusie van gehydrolyseerd caseine met pH 2. Dit
resultaat bevestigde het idee, dat de pancreas-secretie an sich niet de oorzaak isvan de
verminderde prestaties van pas-gespeende biggen. De verstoringen in voeropname
(hoeveelheid en patroon), die vaak optreden na het spenen, kunnen de geleidelijke
ontwikkeling van de (eiwit)verteringscapaciteit vanjonge biggen belemmeren.
Meer (praktische en wetenschappelijke) aandacht voor voeropname(patronen)
gedurende de eerste week na het spenen zou een bijdrage kunnen leveren aan de
verbetering van de prestaties van pas-gespeende biggen.
Conclusies
De verschillen in schijnbare stikstof verteerbaarheid tussen ondermelkpoeder, soja
produkten envismeel zijnvooral tewijten aanverschillen inendogene stikstof-verliezen.
De exocriene pancreas is niet de belangrijkste bron van endogeen stikstof verlies in
jonge biggen.
Pas-gespeende biggen zijn in staat om hun pancreas-secretie te verhogen in respons op
intraveneuze of intraduodenale stimulatie.
Bijjonge biggen is de voeropname na het spenen belangrijker dan de eiwitbron in het
voer voor de ontwikkeling van pancreas protease activiteiten.
Voersamenstelling kan de voeropname van pas-gespeende biggen bei'nvloeden.
Onvoldoende overeenstemming met betrekking tot monster-bewaaromstandigheden en
enzym-analyses bemoeilijkt de interpretatie van literatuur-gegevens.
164
LIST OF PUBLICATIONS
1. Storage of porcine pancreatic juice: effect on enzyme activities.
C.A. Makkink, L.A.J. van der Westerlaken, L.A. den Hartog, M.J. van Baak, J.
Huisman. Zeitschriftfur Tierphysiologie, Tieremahrung und Futtermittelkunde 63:267272,1990.
2. Pancreatic secretion in pigs.
C.A. Makkink, M.W.A. Verstegen. Zeitschriftfur Tierphysiologie, Tieremahrungund
Futtermittelkunde 64:1290-208, 1990.
3. Enzymtoevoegingen aan varkensvoeders.
C.A. Makkink. De Molenaar 29/30:997-999, 19July 1989
4. Enzymbeimischungen bei Schweinefutter.
C.A. Makkink. Kraftfutter12:482-483, 1989
5. Bewaren van pancreassap ten behoeve van enzymanalyses.
C.A. Makkink, L.A.J. van der Westerlaken, L.A. den Hartog, M.J. van Baak, J.
Huisman. Nederlandstalige Voedingsfysiologendag 7-4-1989, Leuven, pp 62-63.
6. The role of the stomach and the pancreas in the digestion of different protein sources
in the piglet.
C.A. Makkink, B. Kemp, M.W.A. Verstegen, J. Huisman. EAAP-meeting. 8-12
July1990, Toulouse,Frankrijk pp 328-329.
7. Digestion of skimmilk powder and soy protein concentrate in the proximal digestive
tract of young piglets.
C.A. Makkink, P.J.M. Berntsen, B.M.L. Op den Kamp, B. Kemp, M.W.A.
Verstegen. The 6th International Symposium on ProteinMetabolism and Nutrition.
EAAP, 9 -14 June 1991,Heming, Denmark, pp 45-47.
8. Aspekte bei der Fiitterung des Absetzferkeln.
M.W.A. Verstegen, C.A. Makkink, B. Kemp. 11.Intensiv Seminar der Steirischer
Gesundheitsdienst.24 -31 march 1990,Ischl. pp 173-190.
9. Onderzoek naar pancreasenzymsecretie.
C.A. Makkink. Themamorgen 'Antinutritionelefactoren in de veevoeding'. 6
december 1988,Zodiac,Wageningen.
10. Maagdarmstoornissen: Zootechnische oorzaken en maatregelen t.a.v. voeding.
C.A. Makkink. PHLO-cursus 'Varkenshouderij'. mei,juni, oktober1989.
165
11. Some aspects of the digestive processes in the small and large intestine in pigs.
L.A. den Hartog, A.F.B.van der Poel, J. Huisman, C.A. Makkink. Proc.14th
International Cong-ess of Nutrition, Seoul (1989). Volume 1:963-966.
12. Verterings-fysiologisch onderzoek bijjonge biggen.
C.A. Makkink, B. Kemp. Varkens,mei 1990, pl2-13.
13. Evaluation of a method to study protein digestion in the proximal digestive tract of
young piglets.
C.A. Makkink, B. Kemp, J.M. Fentener van Vlissingen.Proc. VthInternational
Symposium on Digestive Physiology in Pigs,Wageningen(Doorwerth), Netherlands,
24-26april1991, EAAP Publicationno 54, 1991.pp 173-178.
14. Endogenous N losses at the terminal ileum of young piglets fed diets based on either
skimmilk powder or soybean meal.
C.A. Makkink, T. Heinz. Proc.VthInternational Symposium on Digestive Physiology
in Pigs,Wageningen(Doorwerth), Netherlands,24-26april1991, EAAP Publication
no 54, 1991.pp 196-200.
15. Effect of peas and pea isolates on protease activities in pancreatic tissue of piglets.
M.P. Le Guen, J. Huisman, C.A. Makkink. Proc. VthInternational Symposium on
Digestive Physiology in Pigs,Wageningen(Doorwerth), Netherlands, 24-26april 1991,
EAAP Publication no 54, 1991.pp 207-210.
16. Determination of trypsin and chymotrypsin acticity in pancreatic juice, tissue and
chyme: the effect of freeze-drying and storage.
M.J. van Baak, E.C. Rietveld, C.A. Makkink. Proc. VthInternational Symposium on
Digestive Physiology in Pigs,Wageningen(Doorwerth), Netherlands,24-26april 1991,
EAAP Publication no 54, 1991.pp 356-360.
17. Endogenous N losses at the terminal ileum of young piglets fed diets based on four
different protein sources.
C.A. Makkink, T. Heinz, M.W.A. Verstegen, W.B. Souffrant. Br.J.Nutr.
(submitted)
18. Effect of dietary protein source on feed intake, growth, pancreatic enzyme activities
and jejunal morphology in newly weaned piglets.
C.A. Makkink, N. Negulescu, Qin Guixin, M.W.A. Verstegen. Kurzfassungender
Vortrage zur 46. Tagung vom 31.Man -2.April 1992in Gottingen.Gesellschaftfur
Ernahrungs-physiologie, 6000Frankfurt/Main,Zimmerweg 16. pp 78-79.
19. Gastric protein breakdown and pancreatic enzyme activities in response to different
dietary protein sources in newly weaned piglets.
C.A. Makkink, P.J.M. Berntsen, B.M.L. Op den Kamp, B. Kemp, M.W.A.
Verstegen./. Anim. Sci. (submitted)
166
20. Effect of dietary protein source on feed intake, growth, pancreatic enzyme activities
and jejunal morphology in newly weaned piglets.
C.A. Makkink, G. P. Negulescu, Qin Guixin, M.W.A. Verstegen. Br.J.Nutr.
(submitted)
21. Verkennende Studie Fundamenteel Dierfysiologisch Onderzoek - onderdeel
Stofwisselingsfysiologie.
J. Dijkstra, C.A. Makkink. NRLO-rapport. 1993.106pp.Inpress.
22.Aspects of feed intake in young piglets.
C.A. Makkink. Pigsjan/feb 1993, pl3,15
23. The effect of the level of isotope infusion and the choice of precursor pool on the
determination of endogenous ileal nitrogen excretion in the young pig using the
16
N-isotope dilution technique.
H. Schulze, C.A. Makkink, P.van Leeuwen, M.W.A. Verstegen. Br.J. Nutr.
(submitted)
24. Factors influencing protein digestive capacity in newly weaned piglets.
C.A. Makkink. International Congress on 'Nitrogen Flowin PigProductionand
Environmental Consequences'. Wageningen(Doorwerth),TheNetherlands.8-11June,
1993.
25. Endogenous nitrogen (N) losses as measured with two independent methods.
H. Schulze, C.A. Makkink, W.B.Souffrant, M.P. le Guen, M.W.A. Verstegen.
International Congress on 'Nitrogen Flowin PigProductionand Environmental
Consequences'. Wageningen(Doorwerth),TheNetherlands.8-11June,1993.
167
CURRICULUMVTTAE
Caroline Annette Makkink werd op 3 September 1962, om 13.30 uur geboren te
Arnhem. Zij behaalde in 1980 het VWO-diploma aan het Stedelijk Gymnasium te
Arnhem. In September van datzelfde jaar begon ze met de studie Zootechniek aan de
toenmalige Landbouw Hogeschool te Wageningen. In maart 1988studeerde zij af met
Veehouderij (Gezondheids- en Ziekteleer) en Dierfysiologie als hoofdvakken en
Veehouderij (Ethologie) als bijvak.
Vanaf 1 april 1988 was zij aangesteld als Assistent in Opleiding bij de vakgroep
Veevoeding, waar het onderzoek beschreven in dit proefschrift werd verricht.
Van 1oktober 1992tot 1februari 1993was zij aangesteld als toegevoegd onderzoeker
bij de vakgroep Veevoeding. In deze periode werd een verkennende studie met
betrekking tot het fundamenteel dierfysiologisch onderzoek (onderdeel stofwisselingsfysiologie) in Nederland voltooid in opdracht van de Nationale Raad voor Landbouwkundig Onderzoek.
Op 29januari 1993 trouwde zij met Ben Rankenberg.
Per 1 maart 1993 is zij in tijdelijke dienst aangesteld als onderzoeker klimaat bij het
Proefstation voor de Varkenshouderij te Rosmalen.
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